Methods for Treating GI Syndrome and Graft versus Host Disease

ABSTRACT

We have discovered that administering anti-ceramide antibody treats and prevents an array of diseases mediated by cytolytic T lymphocyte (CTLs)-induced killing and by damage to endothelial microvasculture, including radiation-induced GI syndrome, Graft vs. Host diseases, inflammatory diseases and autoimmune diseases. We have also discovered new anti-ceramide monoclonal antibodies, that have therapeutic use preferably in humanized form to treat or prevent these diseases.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of Provisional Appln. 60/916,298, filedMay 6, 2007, the entire contents of which are hereby incorporated byreference as if fully set forth herein, under 35 U.S.C. §119(e).

STATEMENT OF GOVERNMENTAL INTEREST

This invention was made with Government support under NationalInstitutes of Health Grants CA85704. The Government has certain rightsin the invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention is in the field of methods for treating and preventing GISyndrome and Graft Versus Host Disease.

2. Description of the Related Art

Radiation remains one of the most effective treatments for a widevariety of malignant cells; however healthy cells of the bone marrow,hair follicle, epidermis and gastrointestinal tract are extremelysensitive to radiation-induced cell death, limiting the effective use ofthis therapy for the treatment of cancer. Bone marrow transplantation isanother way to treat advanced cancer, however, organ transplantsfrequently evoke a variety of immune responses in the host, whichresults in rejection of the graft and graft-versus-host disease(hereinafter, referred to as “GVHD”). Bone marrow transplantation iscurrently used to treat a number of malignant and non-malignant diseasesincluding acute and chronic leukemias, myelomas, solid tumors (R. J.Jones, Curr Opin Oncol 3 (2), 234 (1991); G. L. Phillips, Prog Clin BiolRes 354B, 171 (1990)), aplastic anemias and severe immunodeficiency's(R. P. Gale, R. E. Champlin, S. A. Feig et at, Ann Intern Med 95 (4),477 (1981); G. M. Silber, J. A. Winkelstein, R. C. Moen et at, ClinImmunol Immunopathol 44 (3), 317 (1987)). The conditioning regimenrequired prior to transplantation, designed to ablate or suppress thepatient's immune system, renders the patient susceptible to neoplasticrelapse or infection. Recent use of unrelated and HLA non-identicaldonors has unfortunately increased the incidence of GvHD. While removalof T cells from the donor marrow graft ameliorates GvHD, this strategyincreases graft failure rates and markedly diminishes thetherapeutically-beneficial graft-versus-tumor effect. As such, overallsurvival does not improve. Further, despite strong pre-clinical data,attempts to improve GvHD outcomes by diminishing inflammatory cytokineaction by adding TNF antagonists to corticosteroids, the standard ofcare for acute GvHD, has provided limited therapeutic benefit. Thusthere is an urgent need for alternative strategies to reduce theincidence and severity of GI syndrome and GvHD, if it is to be optimizedclinically.

DESCRIPTION OF THE DRAWINGS

FIG. 1. ASMase and Bax deficiency protect C57BL/6 intestinal mucosaagainst radiation-induced microvascular endothelial apoptosis. Proximaljejunal specimens were obtained at 4 hours after 15 Gy TBI of wild type(second panel) and asmase^(−/−) (third panel) and Bax^(−/−) (fourthpanel) C57BL/6 mice, and compared with a specimen obtained from anunirradiated wild type mouse (first panel). Apoptotic nuclei ofendothelial cells (red, CD31 stain) were identified in the villus laminapropria by TUNEL staining as condensed or fragmented brown nucleicontrasting with the blue stain of non-apoptotic nuclei. Arrows indicateapoptotic endothelial cells.

FIG. 2 ASMase deficiency protects intestinal mucosa againstradiation-induced microvascular endothelial apoptosis and crypt stemcell lethality. (A) Frequency histograms of apoptotic cells in thevillus lamina propria of irradiated asmase^(+/+) and asmase^(−/−) miceat 4 hours after 0 to 15 Gy TBI assessed by TUNEL staining. Apoptoticcells were scored in the lamina propria of 200 villae per point. Datarepresent mean scores from two experiments. (B) Transverse sections ofC3HeB/FeJ proximal jejunum were obtained either before irradiation or at3.5 days after irradiation and stained with hematoxylin. Largehyperchromatic crypts are seen in the irradiated specimens (middle andlower panels), typical for surviving regenerating crypts, which aresignificantly enlarged as compared to control unirradiated crypts (upperpanel). (C) H&E-stained proximal jejunum and femur sections obtainedfrom autopsy of asmase^(+/+) and asmase^(−/−) C57BL/6 following 15 GyTBI. The day of lethality is noted.

FIG. 3 Bax deficiency protects mouse intestines againstradiation-induced endothelial apoptosis, crypt lethality and the lethalGI syndrome. (A). C57B1/6 mice were exposed to 15 Gy TBI (A) or 13-15 Gy(B) and tissue samples were obtained and processed as described in FIGS.1 and 2 Similar results were obtained in two experiments for eachresponse shown. Data are reported as (A) mean apoptotic cells in thelamina propria of 200 villae per point and (B) mean±SEM surviving cryptsfrom 10-20 circumferences scored for each of 4 mice. (C) Actuarialsurvival curves of 8-12 week-old C57BL/6 mice receiving autologous bonemarrow transplantation following exposure to 12-15Gy TBI. Actuarialsurvival was calculated by the product limit Kaplan-Meier method[Kaplan, 1958 #47]. 4-10 animals were irradiated per group. Datarepresent collated survival results from multiple experiments.

FIG. 4 Neutralization of Ceramide Antagonizes Platform Generation,Attenuating Radiation-induced Apoptosis in vitro. (A) Pre-incubation ofJurkat T cells with anti-ceramide MID 15B4 (1 microgram/ml) 15 min priorto 10 Gy IR attenuated platform generation. Platforms were quantified bybright field microscopy following staining with anti-ceramide MID15B4(1:30) and Texas-Red-conjugated anti-mouse IgM (1:500). (B) Apoptosiswas quantified in Jurkat cells by nuclear morphologic analysis followingHoeschst bisbenzimide staining with or without preincubation withanti-ceramide MID15B4 (1 microgram/ml). Data are derived from minimum150 cells obtained from three independent experiments.

FIG. 5 Sequestration of ceramide protects C57BL/6 intestinal mucosaagainst radiation-induced microvascular endothelial apoptosis, cryptstem cell death and lethal GI toxicity. (A) Crypt survival assessed bythe crypt microcolony assay. Surviving crypt were identified as shown inFIG. 4 and counted. Data for computation of the surviving fraction ateach dose level was compiled from 2-4 animals irradiated concomitantly,with 10-20 circumferences scored per mouse. Data are reported asmean±SEM. (B) Frequency histograms of apoptotic cells in the villuslamina propria of irradiated asmase^(+/+) and asmase^(−/−) mice 4 hoursafter 15 Gy TBI assessed by TUNEL staining. Apoptotic cells were scoredin the lamina propria of 200 villae per point. Data represent collatedmean scores from two experiments. (C) Actuarial survival curves of 8-12week-old C57BL/6 mice administered anti-ceramide or IgM and exposed to15 Gy TBI. Actuarial survival was calculated by the product limitKaplan-Meier method [Kaplan, 1958 #47]. 5-10 animals were irradiated pergroup. Similar data were observed in 3 experiments. (D)) Pretreatment ofC57BL/6 mice with anti-ceramide MID 15B4 (100 micrograms) 15 min priorto 15 Gy TBI attenuated endothelial apoptosis compared to control IgMtreatments. Small intestine and lung tissue obtained 4 hrs following 15Gy-irradiation were stained by TUNEL. Apoptotic cells are indicated bybrown-stained nuclei. Data (mean±SEM) were obtained from minimum 150villi from two independent experiments.

FIG. 6 Flow chart showing the strategy used to generate humanizableanti-ceramide monoclonal antibody.

FIG. 7 Development of antigen (Ag), Validation of ELISA for Screening.(Inset) BSA-conjugated ceramide was generated by synthesizingBSA-conjugated C₁₆ fatty acid onto a sphingoid base. Validation of theAg for antibody screening was performed by ELISA assay, in whichdecreasing amounts of Ag were fixed to a plate, and following blockingeach well was incubated with anti-ceramide MID15B4 antibody (1:100)followed by horseradish peroxidase-conjugated anti-mouse IgM. OD wasassessed following administration of (horseradish peroxidase) HRPsubstrate at 650 nm

FIG. 8 BSA-ceramide ELISA identified enhanced binding activity insupernatant #3673 following immunization with Kaposi sarcoma cells.Binding of plasma samples obtained from immunized mice by ELISA at 1:100dilution identified higher binding of ceramide by sample #3673 vs.#3674. Binding activity remained following immortalization of antibodyproducing B cells (sn73-I-C6), enabling the isolation of monoclonal 2A2IgM with anti-ceramide binding activity (not shown).

FIG. 9 Purified monoclonal 2A2 antibody binds to BSA-ceramide. Elisarevealed that 2A2 mouse monoclonal IgM binds to BSA-ceramide. Elisashows significantly more binding capacity of 2A2 vs. control IgM,performed as in FIG. 7. 2A2 binds ceramide 5-10× less efficiently thanMID15B4 mouse IgM.

FIG. 10 2A2 antagonizes radiation-induced apoptosis in vitro.Pre-incubation of Jurkat T cells with anti-ceramide 2A2 (0-100microgram/ml) 15 min prior to 8 Gy IR. Apoptosis was quantified inJurkat cells by nuclear morphologic analysis following Hoeschstbisbenzimide staining with or without preincubation with anti-ceramideantibody. Data are derived from minimum 150 cells obtained from threeindependent experiments

FIG. 11 2A2 Enhanced Crypt Survival Following 15 Gy in vivo. (A)Pretreatment of C57BL/6 mice with increasing doses of 2A2 anti-ceramide(0-750 micrograms) improves crypt survival 3.5 d following 15 Gy TBI.(B) 2A2 anti-ceramide antibody increases crypt survival following 8-15Gy total body irradiation by a dose-modifying factor (DMF) of 1.2 (inprevious studies, ASMase deficiency increased crypt survival by a DMF of1.2 in C57BL/6 mice). Crypt survival was determined as in FIG. 5 c.

FIG. 12 2A2 antibody improves survival of C57BL/6 mice exposed to 14-17Gy single-dose radiation. C57BL/6 mice were irradiated with 14-17 Gy TBIwith or without 750 micrograms 2A2 15 min prior to IR. Mice were infusedwith 3×10⁶ autologous bone marrow cells within 16 hour of IR. Survivalwas monitored and expressed via Kaplan-Meier parameters. Statisticalsignificance (P<0.05) was achieved at each dose.

FIG. 13 2A2 Antibody Attenuates Radiation-induced GI death in vivo,recapitulating the asmase^(−/−) phenotype. Necropsy results of micesacrificed when moribund from survival studies performed in FIG. 12. GIdeath was assessed when proximal jejunum specimen appear >90% denuded ofcrypt-villi units and crypt regeneration is absent. Bone marrow (BM)death was assessed when decalcified femur sections reveal depletion ofhematopoietic elements and massive hemorrhage.

FIG. 14 A cartoon illustrating the immunopathophysiology of acute GvHD.

FIG. 15 Host ASMase regulates graft-vs.-host-associated morbidity andmortality. Lethally-irradiated (1100 cGy) C57BL/6^(asmase+/+) orC57BL/6^(asmase−/−) mice received intravenous injection of LP TCD-BMcells (5×10⁶) with or without splenic T cells (3×10⁶). Kaplan-Meiersurvival (A) and clinical GvHD score;¹¹⁷ (B) derived from weeklyassessment of 5 clinical parameters (weight loss, hunched posture,decreased activity, fur ruffling, and skin lesions) are shownrepresenting 6-8 BM control and 13-14 BM+T cell recipients per groupcompiled from two experiments. Statistical analysis is as follows: (A) □vs. ▪ p<0.001, ▪ vs.  p<0.001. (B) □ vs. ▪ p<0.05, ▪ vs.  p<0.05.

FIG. 16 In vivo activated allogeneic CTLs require target hepatocyteASMase for efficient killing ex vivo. Hepatocytes, isolated as describedin Example 1, were coincubated with splenic T cells harvested fromlethally-irradiated wild type C57BL/6 recipients 10-14 days followingtransplantation of LP BM+T cells. (A) 2×10⁶ GvH-activated CTLs werecoincubated with 0.5×10⁶ wild type C57BL/6 or B6.MRL.lpr (FasR^(−/−))hepatocytes (left panel) in complete medium for 16 hr. Alternatively,DMSO or concanamycin A pre-treated (100 ng/ml, 30 min) GvH-activatedCTLs were coincubated with 0.5×10⁶ wild type C57BL/6 hepatocytes for 16hr (right panel). Apoptosis was quantified following fixation by nuclearbisbenzimide staining. (B) asmase hepatocytes are resistant to apoptosisinduced by GvH-activated CTLs. CTL coincubation was performed as in (A)and apoptosis was quantified 16 hr thereafter. (C) Representative imagesof asmase^(+/+) (top left panel) and asmase^(−/−) (bottom left panel)C57BL/6 hepatocytes following 10 min coincubation in suspension with2×10⁶ GvH-activated T cells. Hepatocytes were fixed and stained withDAPI and Cy-3-labelled anti-ceramide mAb as described in Example 1.Arrows indicate ceramide-rich platform generation on the outer leafletof the plasma membrane. Note that after incubation, cells werecentrifuged at 50×g for 4 min at 4° C. prior to staining and imaging.Hence CTLs (small blue nuclei) distributed with hepatocytes (large bluenuclei) do not reflect biologic association. (D) Quantification ofceramide-rich platforms in asmase^(+/+) and asmase hepatocytes followingincubation with 2×10⁶ GvH-activated CTLs. 0.5×10⁶ hepatocytes werecoincubated for the indicated times, fixed and stained as above. (E)Exogenous C₁₆-ceramide bypasses the requirement for target cell ASMase,conferring apoptosis onto GvH-activated CTL-stimulated asmasehepatocytes. Apoptosis was quantified as in (A). (F) Disruption ofmembrane GEMs with nystatin inhibits CTL-induced hepatocyte apoptosis.0.5×10⁶ wild type hepatocytes, preincubated with 50 μg/mL nystatin for30 min and resuspended in RPMI containing 1% lipid-free FBS, werecoincubated with 2×10⁶ GvH-activated T cells and apoptosis wasquantified as in (A). Data (mean±SEM) represent triplicatedeterminations from three independent experiments each for panels A, B,D, E and F.

FIG. 17 Schematic of the in vitro mixed lymphocyte reaction (MLR) assay.

FIG. 18 Schematic of the in vitro activation-induced cell death (AICD)assay.

FIG. 19 In vitro activated CTLs require target splenocyte ASMase forefficient killing. (A) Representative images of ceramide-rich platforms(arrows) formed on the surface of Mitotracker Red-labeled,conA-activated (5 mg/ml for 24 hr) target C57BL/6^(asmase+/+) andC57BL/6^(asmase−/−) splenocytes, upon coincubation for 20 min witheffector Balb/c splenic T cells that had been activated in vitro with2×10⁶ irradiated (2 Gy) C57BL/6 splenocytes/ml media for 5 days at atarget:effector ratio of 2:1. Target splenocytes were fixed with 4%formalin-buffered phosphate, and stained with DAPI and FITC-labeledanti-ceramide mAb. (B) Lysis of ⁵¹Cr-labelled target C57BL/6^(asmase+/+)and C57BL/6^(asmase−/−) splenocytes following coincubation with effectorBalb/c splenic T cells for 6 hr measured by the chromium-release assay.(C) Cytolytic response of ⁵¹Cr-labelled target C57BL/6^(asmase−/−)splenocytes to activated effector Balb/c splenic T cells as in (B), inthe presence of 500 nM C₁₆-ceramide or C₁₆-dihydroceramide (DCer). (D)Representative images of ceramide-rich platforms (arrows) formed on thesurface of C57BL/6^(asmase+/+) and C57BL/6^(asmase−/−) C57BL/6 splenic Tcells 4 hr following induction of AICD with 10 ng/ml anti-CD3 asdescribed in Materials and Methods. Cells were fixed with 4%formalin-buffered phosphate, and stained with DAPI and FITC-labeledanti-ceramide mAb. AICD induces a 2.0±0.1 fold increase in the overallceramide signal as determined by mean fluorescence intensity inasmase^(+/+) T cells (p<0.005 compared to unstipulated controls), notevident in asmase^(−/−) T cells, accounting for the difference inoverall staining between panels. (E) Confocal microscopic detection ofceramide (second panels from top) and GM₁ (third panels from top)colocalization in platforms following AICD induction as in (D).Platforms were identified using Cy3-anti-mouse IgM detection ofanti-ceramide MID15B4 and FITC-conjugated cholera toxin, respectively.(F) Apoptotic response of C57BL/6^(asmase+/+) or C57BL/6^(asmase−/−)splenic T cells after AICD apoptotic fratricide was induced as in (D).Apoptosis was quantified 16 hr thereafter following nuclear bisbenzimidestaining. (G) AICD was initiated in C57BL/6^(asmase−/−) splenic T cellsas in (D), in the presence of 500 nM C₁₆-ceramide orC₁₆-dihydroceramide. Apoptosis was quantified 16 hr thereafter followingnuclear bisbenzimide staining. Data (mean±SEM) represent triplicatesamples from three independent experiments for panels B, C, F, and G.

FIG. 20 2A2 Antibody Impacts acute GvHD in vivo, partiallyrecapitulating the asmase^(−/−) phenotype. Kaplan-Meier survivalanalysis following transplantation of LP BM and T cells as described inFIG. 14. The group receiving 2A2 antibody received 750 microgramsantibody 15 min prior to the first half of 1100 cGy split-dose TBI.

FIG. 21 2A2 Antibody Attenuates Serum Cytokine Storm Associated withacute GvHD. Serum was harvested on day 7 following BMT from miceundergoing experimental acute GvHD from FIG. 15. Serum interferon-gammawas quantified by ELISA, according to manufacturer's protocol (R&DSystems).

FIG. 22 Host ASMase determines graft-vs.-host target organ injury andapoptosis. C57BL/6^(asmase+/+) or C57BL/6^(asmase−/−) mice receivedtransplants and were sacrificed 21 days thereafter for histopathologicanalysis. (A) Representative 5 μM H&E-stained liver sections showingincreased lymphocyte infiltration, portal tract expansion andendotheliitis in asmase^(+/+) hosts receiving allogeneic T cellscompared to asmase^(−/−) littermates. (B) Representative 5 μMTUNEL-stained sections of proximal jejunal crypts and villi displaylamina propria and crypt apoptosis. Arrows indicate cells containingcondensed or fragmented brown nuclei contrasting with the blue stain ofnon-apoptotic nuclei, quantified in (C) and (D), respectively. Frequencyhistograms of apoptotic cells in the villus lamina propria (C) representdata from 150 villae per point, collated from 2 experiments. Cryptapoptosis (D) was scored in 200 crypts per point, and represent mean±SEMcollated from 2 experiments. (E) C57BL/6 recipient hosts received marrowtransplants as above, or alternatively, 10×10⁶ TCD-BM cells with orwithout 0.5×10⁶ T cells from B10.BR (H2^(k)) donors. Skin (tongue andear) were harvested 14 (B10.BR recipients) or 21 (LP) days post marrowtransplant and GvHD score was determined by the number of dyskeratoticand apoptotic keratinocytes per millimeter of epidermis (mean±SEM) asassessed in blinded fashion on H&E-stained sections. Data represent 4-14mice per group compiled from three independent experiments.

FIG. 23. Donor CD8⁺ T cell expansion are impaired in asmase^(−/−) hosts.(A) C57BL/6^(asmase+/+) and C57BL/6^(asmase−/−) recipients were infusedwith 15-20⁶ CFSE-stained splenic CD3⁺ T cells from LP/J donors asdescribed in Example 1. Spleens were harvested 72 hrs thereafter andmulticolor flow cytometry was performed. Percentage of CFSE “high”(cells with mean fluorescent values >10³) and “low” (mean fluorescentvalues <10³) CD4⁺ and CD8⁺ populations are shown from one representativeof two independent experiments. (B) Flow cytometric analysis of Ly9.1⁺donor LP/J T cells harvested from C57BL/6 asmase^(+/+) and asmaserecipients 14 days following LP/J BM and T cell infusion, as describedin Example 1. Data (mean±SEM) represent 4-12 determinations from twoindependent experiments.

FIG. 24 T cell proliferative capacity remains intact in asmase^(−/−)hosts. Thymidine incorporation assay measuring proliferation of splenicT cells harvested from C57BL/6^(asmase+/+) or C57BL/6^(asmase−/−)recipients of donor LP BM and T cells in response to syngeneic (LP) orallogeneic (Balb/c) splenocytes or mitogen (ConA). Data (mean±SEM)represent triplicate determinations from three independent experiments

FIG. 25 1H4, 5H9 and 15D9 hybridoma cell lines were established aftersix subclonings. Hybridoma supernatants were screened in a Jurkat cellapoptosis inhibition assay and showed protective effects that are dosedependent and comparable to 2A2. The isotypes of the three antibodieshave been established. 15D9 mAb is IgM, kappa. 1H4 and 5H9 mAbs aremIgG3, kappa.

LIST OF ABBREVIATIONS

-   ASMase—acid sphingomyelinase-   BMT—bone marrow transplant-   CTLs—cytotoxic T lymphocytes-   ERK—extracellular signal-related kinase-   FADD—Fas-associated death domain-   FcRγII—Fc receptor γII-   FITC—fluorescein isothiocyanate-   GVHD—Graft-Versus-Host-Disease-   GVT—Graft-Versus-Tumor-   IL—interleukin-   JNK—c-Jun N-terminal kinase-   mHAg(s)—minor histocompatability antigen(s)-   MHC—major histocompatability complex-   MLR—mixed lymphocyte reaction-   SDS-PAGE—sodium dodecyl sulfate-polyacrylamide gel electrophoresis-   TCR—T cell receptor-   TNF—tumor necrosis factor-   TUNEL—terminal dUTP nick-end labeling

DETAILED DESCRIPTION

We have discovered that administering anti-ceramide antibody treats andprevents an array of diseases mediated by cytolytic T lymphocyte(CTLs)-induced killing and by damage to endothelial microvasculture,including radiation-induced GI syndrome, Graft vs Host diseases,inflammatory diseases and autoimmune diseases (herein the enumerateddiseases). We have also discovered a new anti-ceramide monoclonalantibody 2A2 and others described below, that have therapeutic usepreferably in humanized form to treat or prevent the enumerateddiseases. Other embodiments of the invention are directed to combinationtherapy to treat or prevent the above enumerated diseases byadministering anti-ceramide antibody together with one or more statins;or with imipramine or other ASMase inhibitor or Bax inhibitor. Yet otherembodiments include administering an antisense nucleotides or smallinterfering RNA in an amount that reduces expression of ASMase, Bax andBak or otherwise reduces or ameliorates a symptom of any of theenumerated diseases. Finally, certain embodiments are directed tocompositions for therapeutic use in treating or preventing theenumerated diseases that include an anti-ceramide antibody and one ormore statins or imipramine.

Extracellular Ceramide is Required for Radiation-Induced Apoptosis

Lipid rafts, which are distinct plasma membrane microdomains comprisedof cholesterol tightly packed with sphingolipids, in particularsphingomyelin, creating a liquid-ordered domain within theliquid-disordered bulk plasma membrane. Rafts differ in their proteinand lipid composition from the surrounding membrane, housing signalingmolecules including multiple glycosylphosphatidylinositol (GPI)-anchoredproteins, doubly-acylated tyrosine kinases of the Src family andtransmembrane proteins. In addition, rafts serve as sites that multiplereceptors translocate into or out of upon their activation, includingthe B cell receptor (BCR) upon encountering antigen. Recent evidencesuggests that these translocation events are crucial for multiple signaltransduction cascades.

Sphingolipids, which were classically viewed as structural components ofcell membranes, were discovered to be important regulators of signaltransduction by the determination that 1,2-diacylglycerols stimulatedsphingomyelin hydrolysis to ceramide⁵ By identifying activation of anacidic sphingomyelinase (ASMase) that generates ceramide in GH3pituitary cells, these studies introduced a potential role for ceramideas a second messenger. This role was supported by the identification ofmodulated protein phosphorylation events and kinase activity in vitro byaddition of exogenous ceramides (R. N. Kolesnick and M. R. Hemer, J BiolChem 265 (31), 18803 (1990), S. Mathias, K. A. Dressler, and R. N.Kolesnick, Proc Natl Acad Sci USA 88 (22), 10009 (1991)). Ultimately,the coupling of tumor necrosis factor receptor (TNFR) activation withceramide generation in a cell free system, antagonism of TNF-α-mediatedsignaling by inhibition of ceramide generation, and the recapitulationby exogenous ceramides of TNF-α signaling in cells devoid of ceramidegeneration established ceramide as a bona fide lipid second messenger(K. A. Dressler, S. Mathias, and R. N. Kolesnick, Science 255 (5052),1715 (1992)).

Acid sphingomyelinase (ASMase) is a sphingomyelin-specific phospholipaseC (sphingomyelin phosphodiesterase) that exists in two forms, alysosomal and secretory form; it initiates a rapid stress response inmany cell types (R. Kolesnick, Mol Chem Neuropathol 21 (2-3), 287(1994); J. Lozano, S. Menendez, A. Morales et al., J Biol Chem 276 (1),442 (2001); Y. Morita, G. I. Perez, F. Paris et al., Nat Med 6 (10),1109 (2000); F. Paris, Z. Fuks, A. Kang et al., Science 293 (5528), 293(2001); Santana, L. A. Pena, A. Haimovitz-Friedman et al., Cell 86 (2),189 (1996)). Our recent work showed that clustering of plasma membranelipid rafts into ceramide-enriched platforms amplify stimuli capable ofactivating ASMase. Grassme et al., J Biol Chem. 2001, 276:20589,incorporated herein by reference. In these studies we showed that invitro and in vivo, extracellularly orientated ceramide, released uponCD95-triggered translocation of ASMase to the plasma membrane outersurface, enabled clustering of CD95 in sphingolipid-rich membrane raftsand induced apoptosis in Jerkat cells. Whereas ASMase deficiency,destruction of rafts, or neutralization of surface ceramide preventedCD95 clustering and apoptosis, natural ceramide rescued ASMase deficientcells. The data showed that CD95-mediated clustering by ceramide is aprerequisite for signaling and apoptotic cell death. Jurkat cells are ahuman T cell leukemia cell line.

Others have shown that ceramide is required for Fas-induced apoptosis insome cell types. We looked at the requirement for UV-C-induced ceramidegeneration in initiating the apoptotic response. By UV-C is meantultraviolet radiation in the 100-280 nm wavelength range. In thesestudies imipramine was used to inhibit ASMase-activity. Pre-treatment ofJurkat cells with 50 mM imipramine for 30 min decreased baseline ASMaseactivity, abrogated UV-C and Fas-induced ASMase activation at 1 min poststimulation and ceramide generation at 2 minutes, and attenuatedapoptosis at 4 hours post-stimulation. These data showed that ASMaseactivation is indispensable for optimal Fas- or UV-C-induced apoptosis,though they do not define the role of ceramide per se in this response.Grassme et al., J Biol Chem. 2001, 276:20589, incorporated herein byreference.

Ceramide has important roles in differentiation, proliferation andgrowth arrest, but the most prominent role for ceramide is in theinduction of programmed cell death. Exogenous C₈ ceramide andsphingomyelinase mimicked TNF-α-induced DNA fragmentation and loss ofclonogenicity in HL60 human leukemia cells, suggesting that ceramide wasan essential component of apoptotic signaling (K. A. Dressler, S.Mathias, and R. N. Kolesnick, Science 255 (5052), 1715 (1992). Ionizingradiation (IR) stimulates sphingomyelin hydrolysis to ceramide, andexogenous ceramide was able to bypass phorbol ester-mediated inhibitionof radiation-induced ceramide generation and apoptosis (A.Haimovitz-Friedman, C. C. Kan, D. Ehleiter et al., J Exp Med 180 (2),525 (1994)). Lymphoblasts derived from Niemann-Pick patients, a geneticdisease characterized by an inherited deficiency in ASMase activity,proved in a genetic model that ASMase-mediated ceramide generation isrequired for radiation-induced apoptosis, and the development of anASMase-deficient mouse allowed the identification of cell-type specificroles for ceramide (J. Lozano, S. Menendez, A. Morales et al., J BiolChem 276 (1), 442 (2001); P. Santana, L. A. Pena, A. Haimovitz-Friedmanet al., Cell 86 (2), 189 (1996)). Ceramide generation has since beenidentified as requisite for multiple cytokine-, virus/pathogen-,environmental stress-, and chemotherapeutic-induced apoptotic events.Verheij M, et al. Requirement for ceramide-initiated SAPK/JNK signalingin stress-induced apoptosis. Nature. 1996 Mar. 7; 380(6569):75-9;Riethmüller J, et al., Membrane rafts in host-pathogen interactions,Biochim Biophys Acta. 2006 December: 1758(12):2139-47, incorporatedherein by reference.

An emerging body of evidence has since recognized ceramide-mediated raftclustering as sites of signal transduction for bacteria and pathogeninternalization, and radiation- and chemotherapeutic-induced apoptosis.D. A. Brown and E. London, Annu Rev Cell Dev Biol 14, 1 1 1 (1998): J.C. Fanzo, M. P. Lynch, H. Phee et al., Cancer Biol Ther 2 (4), 392(2003); S. Lacour, A. Hammann, S. Grazide et al., Cancer Res 64 (10),3593 (2004); Semac, C. Palomba, K. Kulangara et al., Cancer Res 63 (2),534 (2003; A. B. Abdel Shakor, K. Kwiatkowska, and A. Sobota, J BiolChem 279 (35), 36778 (2004); H. Grassme, V. Jendrossek, J. Bock et al.,J Immunol 168 (1), 298 (2002); M. S. Cragg, S. M. Morgan, H. T. Chan etal., Blood 101 (3), 1045 (2003); ⁶ D. Scheel-Toellner, K. Wang, L. K.Assi et al., Biochem Soc Trans 32 (Pt 5), 679 (2004).; D. Delmas, C.Rebe, S. Lacour et al., J Biol Chem 278 (42), 41482 (2003).; and C.Bezombes, S. Grazide, C. Garret et al., Blood 104 (4), 1166 (2004).

In this context, ceramide-enriched platforms transmit signals forIR-induced apoptosis of Jurkat cells (Zhang and Kolesnick, unpublishedresults) and bovine aortic endothelial cells (Stancevic and Kolesnick,unpublished results), CD40-induced IL-12 secretion and c-Jun Kinasephosphorylation in JY B cells, P. aeruginosa internalization andactivation of the innate immune response in lung, Rituximab-induced CD20clustering and ERK phosphorylation in Daudi and RL lymphoma cells,FcRγII clustering and phosphorylation in U937 human monocytic cells, andresveratrol-, cisplatin- and reactive oxygen species-induced apoptosisin HT29 human colon carcinoma cells and neutrophils. Despite extensivestudies on the downstream effects of ASMase translocation andactivation, little was known of the initiating events mediating itstranslocation onto the outer plasma membrane until we showed that thereis a capsase-independent pathway that initiates ASMase signaling. J. A.Rotolo et al., J. Biol. Chem. Vol 280, No. 29, Issue of July 15,26425-34 (2005), incorporated herein by reference.

It is important to emphasize that there are multiple pathways in a cellto make ceramide in different compartments. Ceramide generated at thecell surface in rafts by ASMase is different from ceramide inside thecell. The results presented below show for the first time thatASMase-generated cell surface ceramide is responsible for causingradiation-induced GI syndrome through damage to endothelialmicrovasculature (a hallmark of GI syndrome). We further show thatASMase-generated ceramide is required for GVHD caused by T-cell mediatedkilling. Thus ASMase generated ceramide is required for both endothelialmicrovasculature damage and T-cell mediated killing. We have furtherdiscovered that inhibiting or sequestering ceramide generated by ASMaseby administering anti-ceramide antibodies in vivo, reducedradiation-induced damage, and can be sued to treat or prevent GIsyndrome and GvHD. Certain embodiments of the present invention aredirected to pharmacological methods to treat or prevent GVHD and GIsyndrome and other T-cell mediated diseases including autoimmunediseases, by blocking ASMase (for example with imipramine or withantisense nucleic acids) or by inactivating cell surface ceramide (forexample with anti-ceramide antibodies alone or together with statins).

Treatment and Prevention of the Lethal GI Syndrome

The intestinal clonogenic compartment housing stem cells of the smallintestine resides at positions 4-9 from the bottom of the crypt ofLieberkühn, and consists of intestinal pluripotent stem cells anduncommitted progenitor clonogens, herein called stem cell clonogens.This group of cells proliferates and differentiates incessantly,replenishing the physiologic loss of enterocytes and otherdifferentiated epithelial cells from the villus apex, thus maintainingthe anatomical and functional integrity of the mucosa. A completedepletion of this compartment appears required to permanently destroythe crypt-villus unit, while surviving stem cell clonogens, albeit evenone per crypt, are capable of regenerating a fully functional crypt.

Radiation targets both the gastrointestinal microvasculature andproliferating crypt stem cells. Apoptosis of the microvascularendothelium in the villus represents the primary lesion of the GIsyndrome, occurring 4 hours following radiation. Endothelial apoptosisconverts lesions to the crypt clonogens from sublethal to lethal,resulting in loss of regenerative crypts and promoting GI toxicity.Immunohistochemical and labeling studies with [³H]TdR and BrdUrdrevealed that crypt stern cell clonogen death does not occur acutelyafter radiation exposure. Rather, the earliest detectable response is atemporary dose-dependent delay in progression through a late S-phasecheckpoint and mitotic arrest, apparently signaled by radiation-inducedDNA double strand breaks (dsb). In mammalian cells, DNA dsbs activatepathways of DNA damage recognition and repair, and a coordinatedregulation of cell cycle checkpoint activity. The intestinal stem cellmitotic arrest appears to represent a regulated event in this pathway.Consistent with this notion, no significant change in crypt number perintestinal circumference is apparent at this stage although crypt sizeprogressively decreases due to continued normal migration of crypttransit and differentiated cells from the crypt into the epitheliallining of the villus. Resumption of mitotic activity at 36-48 hours isassociated with a rapid depletion of crypt stem cell clonogens andreduction in the crypt number per circumference. The mechanism of stemcell depletion has not been fully established.

The lethality of GI stem cell clonogens is best assessed by the numberof crypts surviving at 3.5 days after radiation exposure, whichdecreases exponentially as the dose increases (C. S. Potten and M.Loeffler, Development 110 (4), 1001 (1990), H. R. Withers, Cancer 28(1), 75 (1971), and J. G. Maj, F. Paris, A. Haimovitz-Friedman et al.,Cancer Res 63, 4338 (2003)). Crypts that contain surviving stem cellsproliferate at an accelerated rate, producing typical regenerativecrypts that split or bud to generate new crypts, until the intestinalmucosa regains a normal architecture. TBI experiments in several mousemodels have demonstrated that the number of surviving crypt stem cellsafter exposure to 8-12 Gy is usually sufficient to support a completerecovery of the mucosa. At higher doses, however, massive stem cellclonogen loss may lead to a near total collapse of the crypt-villussystem, mucosal denudation and animal death from the GI syndrome. Thethreshold dose for inducing the GI death, and the TBI dose producing 50%GI lethality (LD₅₀), appear to be strain-specific. Autopsy studies ofC57BL/6 mice exposed to TBI revealed that 25% of the mice exposed to 14Gy and 100% of those exposed 15 Gy succumbed to the GI syndrome at6.8±0.99 days, predicting an LD₅₀ for GI death between 14 and 15 Gy. Incontrast, the reported LD_(50/6) (the LD₅₀ at day 6, serving as asurrogate marker for GI death) for BALB/c mice is 8.8±0.72 Gy, 11.7±0.22Gy for BDF1 mice, 12.5±0.1 Gy for C3H/He mice, 14.9 Gy (95% confidencelimits 13.9-16.0 Gy) for C3H/SPF mice, and 16.4±1.2 Gy for B6CF1 mice,indicating a strain-specific spectrum in mouse sensitivity to death fromthe GI syndrome. Strain variations in the sensitivity of other organs toradiation, such as the bone marrow and lung have also been reported.

Classically, ionizing radiation (IR) was thought to kill cells by directdamage to genomic DNA, causing genomic instability and resulting inreproductive cell death. Haimovitz-Friedman et al. demonstrated in anuclei-free system that apoptotic signaling can alternately be generatedby the interaction of IR with cellular membranes. Extension of thesestudies by us and described herein revealed that ceramide mediated raftclustering is involved in IR-induced apoptosis and clonogenic celldeath. It has long been accepted that the clonogenic compartment of thegastrointestinal (GI) mucosa is the specific and direct target forradiation in inducing GI damage.

In some of our early work we showed that greater protection againstultra violet radiation with UV-C (5-50 J/m²)- and anti-Fas (1-50 ng/mlCH-11)-induced apoptosis in vitro was obtained by inducing ceramideneutralization with anti-ceramide monoclonal antibody combined withcholesterol depletion induced by nystatin in Jurkat cells, than wasobtained using either agent alone. H. Grassme, H. Schwarz, and E.Gulbins, Biochem Biophys Res Commun 284 (4), 1016 (2001), incorporatedherein by reference. In these studies we showed that preincubation ofJurkat cells with anti-ceramide antibody in combination with nystatininhibited raft clustering 1 min post 50 J/m² UV-C- or 50 ng/ml anti-Fasstimulation. Furthermore, inhibiting raft clustering by anti-ceramideand nystatin combination treatment attenuated UV-C (5-50 J/m²)- andanti-Fas (1-50 ng/ml CH-11)-induced apoptosis 4 hours post stimulation(FIG. 2 d) and enhanced cell viability by 2.46- and 2.42-fold,respectively, 7 days post stimulation with 50 J/m² UV-C or 50 nganti-Fas. Importantly, we also observed that anti-ceramide and nystatinpretreatment yielded an approximate 1 log increase in clonogenic cellsurvival compared to vehicle controls after 5-50 J/m² UV-C or anti-Fasstimulation. Plotting these clonogenic survival data according to thesingle-hit multitarget model revealed that pretreatment withanti-ceramide and nystatin increased the D₀ of the dose response curvefrom 1.6±0.7 J/m² to 3.6±1.1 J/m², indicating significant (p<0.05)protection against the reproductive mode of UV-induced cell death, witha dose modifying value of 2.32 at the 10% survival level. Takentogether, these results showed that ceramide-mediated raft clustering atthe surface of Jurkat cells is obligate for apoptotic transmembranesignal transduction induced by UV-C, and that such protection isbiologically-relevant as evidenced by improved clonogenic survival.

There is evidence for a conditional linkage between crypt stem cellclonogen lethality after single-dose radiation and the early wave ofASMase-mediated apoptosis in the endothelium of the intestinalmicrovascular system. Radiation induces rapid translocation of asecretory non-lysosomal form of ASMase into glycosphingolipid- andcholesterol-enriched rafts in the outer leaflet of the plasma membrane(E. Gulbins and R. Kolesnick, Oncogene 22 (45), 7070 (2003) whereceramide is rapidly generated, coordinating transmembrane signaling ofapoptosis. Endothelial cells are 20-fold enriched in secretory ASMasecompared with other cells in the body, and this cell type isparticularly sensitive to radiation-induced apoptosis in vitro and invivo. Genetic inactivation of ASMase in SV129/C57BL/6 mice, orintravenous treatment of C57BL/6 mice with the endothelial cell survivalfactor bFGF prior to whole body irradiation (TBI), attenuatedradiation-induced endothelial apoptosis of the intestinal microvascularsystem, preserved crypt stem cell clonogens, and protected mice againstlethality from the GI syndrome (F. Paris, Z. Fuks, A. Kang et al.,Science 293 (5528), 293 (2001)). Given that the intestinal endotheliumbut not crypt epithelial cells expressed bFGF receptor transcriptsbefore or after irradiation, vascular dysfunction appeared critical forradiation-induced GI damage.

ASMase- and Bax-Deficiency Protects Against Radiation Damage and GISyndrome

This section describes our experiments showing that both asmase^(−/−)and Bax^(−/−) mice are resistant to endothelial apoptosis occurringwithin 4 hours of total body irradiation (TBI). This allows the repairof sublethal lesions incurred by crypt clonogens, regenerating thecrypt-villus system and abrogating the lethal GI syndrome. Thewell-characterized parameters of the GI syndrome make this system anideal model for study of ceramide targeting pharmaceuticals. Below weshow that anti-ceramide antibody attenuated microvascular apoptosis tothe level similar to that of the asmase_(−/−) genotype, promotingclonogenic crypt stem cell survival and GI regeneration. Anti-ceramideprotected 60% of mice receiving 15 Gy TBI and syngeneic BMT,demonstrating that the microvascular protection is biologicallyrelevant.

Previous studies identified an endothelial-stem cell clonogen linkage inthe GI radiation response of the C57BL/6 mouse strain and itsSV129/C57BL/6 hybrid. Further, these studies characterized the role ofASMase in the GI radiation response in the SV129/C57BL/6 hybrid strain.F. Paris, et al. supra. To examine whether genetic inactivation ofASMase protects C57BL/6 from the vascular component in the intestinalresponse to radiation, several of the features that characterize theSV129/C57BL/6 mice intestinal response to radiation were evaluated inasmase^(+/+) and asmase^(−/−) C57BL/6 mice. Typical histologic examplesof the pattern of endothelial response to radiation in asmase^(+/+) andasmase^(−/−) genotypes in this strain are shown (FIG. 1). Consistentwith published observations on SV129/C57BL/6 mice, wild type C57BL/6mice show extensive endothelial apoptosis at 4 hours after 15Gy TBI (12apoptotic endothelial nuclei in the villus lamina propria; FIG. 1 secondpanel), reduced in the asmase^(−/−) and Bax^(−/−) specimen to 3apoptotic nuclei each (FIG. 1, third and fourth panel, respectively).Only occasional (1-3) apoptotic nuclei were observed in the laminapropria of an unirradiated control asmase^(+/+) (FIG. 1, first panel),asmase^(−/−) or Bax^(−/−) (not shown). Endothelial cell apoptosis wasdetected in the wild type mucosa as early as at 3 hours after exposureto 15Gy and reached a maximum at 4 hours (not shown). FIG. 2A displays afrequency histogram of apoptotic nuclei in the intestinal lamina propriaof C57BL/6 mice at 4 hours after exposure to escalating doses of TBI. Amaximal effect was observed in the asmase^(+/+) mucosa at 15Gy, with 92%of the villae displaying >3 apoptotic nuclei/villus and 52% displayingextensive apoptosis (>10 apoptotic nuclei/villus), compared to ≦3apoptotic nuclei/villus observed in control unirradiated mice (p<0.001;n=200 villae from 2 animals counted for each data point). ASMasedeficiency significantly reduced the overall apoptotic response (>3apoptotic nuclei/villus) to 38% and the frequency of extensive apoptosisto 20% of the total villae (p<0.001 each compared to wild typelittermates, respectively; n=200 villae from 2 animals counted for eachdata point). Thus, the kinetics and dose-dependency of radiation-inducedendothelial cell apoptosis in the C57BL/6 strain, and the requirementfor ASMase, were qualitatively and quantitatively similar to those inthe SV129/C57BL/6 mouse strain¹ although the peak incidence of theapoptotic response occurred 1 hour later than in the SV129/C57BL/6strain.

As reported for SV129/C57BL/6 mice (J. G. Maj, F. Paris, A.Haimovitz-Friedman et al., Cancer Res 63, 4338 (2003)), endothelialapoptosis closely correlated with survival of crypt stern cell clonogensafter TBI. FIG. 2 b shows typical cross sections of C57BL/6 miceproximal jejunum at 3.5 days after exposure to 15Gy TBI. Whenunirradiated, the number of crypts/intestinal circumference in thisstrain was 155±1.1. After exposure to 15Gy, a specimen from aC57BL/6^(asmase+/+) mouse shown contained only 3 surviving regeneratingcrypts, compared with 27 in the specimen obtained from aC57BL/6^(asmase−/−) littermate. ASMase deficiency significantlyincreased the crypt surviving fraction at each dose within the range of10-15Gy (p<0.05). Wild type mice 3.5 days following exposure to 15 GyTBI are nearly completely depleted of functional proximal jejunal crypts(FIG. 2 b, middle panel). Genetic inactivation of ASMase enhanced cryptsurvival as evidenced by increased expression of dark-purple stainedregenerative, hyperchromatic crypts (FIG. 2 b, bottom panel). The doserequired to produce an isoeffect of 10% crypt survival (D₁₀) was14.6±0.9 Gy for wild-type and 16.8±1.8 Gy for the ASMase-deficient mice(p<0.01), indicating a dose-modifying factor (DMF) of 1.15±0.14 for theasmase^(−/−) genotype. This value was not different significantly fromthe DMF reported for protection of irradiated C57BL/6 crypt stem cellclonogens by bFGF⁷.

The stem cell clonogen protection afforded by ASMase deletion alsotranslated into protection against C57BL/6 mouse death from the GIsyndrome after 15Gy TBI, similar to that reported for hybridSV129/C57BL/6^(asmase−/−) mice¹. By contrast p53-deficient C57BL/6 micewere not protected from radiation damage (data not shown.)C57BL/6^(asmase+/+) mice died at 5-6 (mean 5.3±0.2) days after TBI (notshown). Autopsies revealed intestinal damage typical of the GI syndrome(extensive denudation of nearly all the crypts and villae) with onlypartial damage to the bone marrow (regions of hemorrhage and depletionof hematopoietic elements mixed with islands of normal hematopoietictissue; FIG. 2 c left and right middle panels, respectively;). Otherorgans were found intact, except for thymic and lymphatic tissues, andoccasional micro-abscesses or focal hemorrhages in various organs, notconsidered as direct causes of death. In contrast C57BL/6^(asmase−/−)mice died at 7.75±0.12 days (p<0.001 when compared toC57BL/6^(asmase+/+) mice). Autopsies revealed typical characteristics ofbone marrow death (widespread hemorrhage and extensive necrosis of thematrix, with complete depletion of hematopoietic elements; FIG. 2 c,right lower panel). Further, in contrast to the complete destruction ofthe crypt/villus network in C57BL/6^(asmase+/+) mice, the intestinalmucosa of C57BL/6^(asmase−/−) littermates showed extensive regenerativeactivity with hyperplastic, chromophilic crypts covering most of theintestinal surface (FIG. 2 c, left lower panel).

Bax Deficiency Phenocopies Asmase^(−/−) Protection from Radiation Damageand GI Syndrome

To eliminate the possibility that an event other than endothelial cellapoptosis, induced by radiation and regulated by ASMase, might impactthe lethality of radiation-injured stern cell clonogens. Experimentswere carried out with the apoptosis-refractory Bax-deficient C57BL/6mice that express wild-type ASMase and mimic the asmase^(−/−) radiationphenotype in tumor endothelium an in oocytes of young mice. Bax and Bakare prototypical proapoptotic Bcl-2 multidomain proteins. Doubledeletions (homozygous recessive Bax−/− and Bak−/− mutations) werepreviously believed to be required to endow resistance to apoptoticstimuli. Data for Bak-deficient mice is not shown.

The wild type C57BL/6^(Bax+/+) strain used in these experimentsdisplayed no detectable baseline endothelial cell apoptosis, andunderwent a time-dependent increase after exposure to 15Gy TBI thatpeaked at 4 hours (not shown). 85% of the villae contained >3endothelial apoptotic nuclei/villus at 4 hours after 15 Gy, and 38%showed an extensive (>10 apoptotic nuclei/villus) apoptotic response(FIG. 3). Bax deficiency significantly reduced the overallradiation-induced apoptotic response to 45% (p<0.05), and the frequencyof extensive apoptosis to 12% of the villae (p<0.001; n=200 villae from2 animals counted for each data point), mimicking the previouslyreported asmase^(−/−) radiation-response phenotype in C57BL/6 andSV129/C57BL/6. Attenuation of endothelial apoptosis by Bax deficiencyprotected crypt clonogens following exposure to 13, 14 and 15Gy TBI(FIG. 3 b). Surviving crypts in wild type C57BL/6 mice at 3.5 daysfollowing 13, 14 and 15Gy TBI, reputedly a measure of surviving cryptstem cells, was decreased from 152±3 in unirradiated intestinalcircumferences to 20.5±1.3, 10.8±0.6 and 2.3±0.3 respectively (survivingfractions of 13.4±0.9%, 7.0±0.4% and 1.47±0.2%, respectively, n=10-20circumferences each from 4 animals per point). Bax deficiency increasedthe number of surviving crypts following 13, 14 and 15 Gy TBI vs. wildtype controls to 42.3.7±2.0, 27.6±1.6 and 18.2±1.3, respectively(surviving fractions of 27.8±1.4%, 18.2±1.1% and 12.0±0.9%,respectively, P<0.001 vs. wild type C57BL/6, FIG. 3 b). The fraction ofsurviving crypts in C57B1/6^(Bax−/−) mice following 13-15 Gy exceededthe 8.5±0.1% surviving crypts reported necessary to support mucosalrecovery and prevent GI death in wild type C57B1/6 mice treated with12Gy alone^(62,71). These data are consistent with the notion of alinkage between endothelial apoptosis and survival of crypt stem cellclonogens after radiation exposure. J. A. Rotolo, et al, Int. J.Radiation Oncology Biol. Phys., Vol. 70, No. 3, 804-815(2008),incorporated herein by reference.

The stem cell clonogen protection provided by Bax deletion wasassociated with protection against mouse death from the GI syndrome,similar to that reported for SV129/C57BL/6 mice¹. Autologous bone marrowtransplantation (BMT) protected 100% of C57B1/6^(Bax+/+) andC57B1/6^(Bax−/−) mice exposed to 13 Gy TBI from BM death (FIG. 3 c).Control animals exposed to these TBI doses but not receiving BMTsuccumbed to BM death with fully repaired (12 Gy) or near completelyrepaired (13 Gy) GI mucosa (Table 1). Table 1 shows that geneticinactivation of Bax or pharmacologic antagonism of ceramide byanti-ceramide antibody inhibits the lethal GI syndrome. Autopsies oftotal body irradiated C57BL/6 mice of Bax^(+/+) or Bax^(−/−) genotypeand C57BL/6 mice administered anti-ceramide antibody or irrelevant IgMcontrol revealed Bax and ceramide signaling are required for the lethalGI syndrome. BM and GI lethality was assessed by histopathologicexamination of H&E stained, 5 μm sections of proximal jejunum and femur.GI lethality was characterized by complete denudation of villus andcrypts, and BM lethality was characterized by depletion of hematopoieticelements from the BM cavity and massive hemorrhage. * denotesaccelerated BM aplasia and mixed BM and GI death.

TABLE 1 Lethal Syndrome Genotype TBI BM GI C57BL/6^(Bax+/+) 12 Gy 100% —13 Gy 100% — 14 Gy  90%  10% 15 Gy — 100% C57BL/6^(Bax−/−) 12 Gy 100% —13 Gy 100% — 14 Gy 100%* — 15 Gy 100%* — C57BL/6 + IgM 15 Gy — 100%C57BL/6 + anti- 15 Gy 100% — ceramide

A switch to death from the GI syndrome occurred in C57B1/6^(Bax+/+) miceat 14 Gy TBI, with 90% of BMT-untreated mice succumbing to this mode ofdeath at 7.7±0.8 days (FIG. 5 c; Table 1). Thus, the C57BL/6 substrainharboring the Bax^(−/−) genotype exhibited an approximate 1Gy increasein GI radiosensitivity compared to previously reported data for awild-type C57BL/6 mouse colony (F. Paris, Z. Fuks, A. Kang et al.,Science 293 (5528), 293 (2001)). Bax deficiency protected from GI deathafter 14 Gy TBI (FIG. 5 c, Table 1), with autopsies showinghyperplastic, chromophilic crypts covering most of the intestinalsurface, indicative of advanced regeneration of the intestinal mucosa,and typical changes of BM death. These findings are consistent with thelevels of crypt stem cell clonogen survival in C57B1/6^(Bax−/−) micereported above (FIG. 3 b) and the observation of a regeneratingintestinal mucosa is probably associated with prolongation of survivalof these mice (8.4±0.5 days; Table 1), which presumably enabledinitiation of mucosal recovery. Autologous BMT permanently rescued 60%of C57B1/6^(Bax−/−) mice exposed to 14 Gy TBI (FIG. 3 c; p<0.05), whilethe remaining animals failed engraftment and succumbed to bone marrowaplasia (Table 1). When the dose was escalated to l5Gy TBI,C57B1/6^(Bax+/+) mice died at 5.5±0.4 days from autopsy-proven mixed GIand BM death (Table 1) and Bax deficiency failed to rescue the animals,despite the apparent availability of sufficient numbers of survivingcrypts (FIG. 3 b) as required for successful recovery of the GI mucosa.The latter phenomenon likely resulted from an accelerated development ofBM aplasia and BM death, due to reasons uncertain, which occurred beforemucosal regeneration became apparent. Consistent with this notion,autologous BMT into 15 Gy TBI-treated C57B1/6^(Bax−/−) mice extendedmice survival to 9.0±0.0 days (FIG. 3 c; p<0.05), and although the levelof engraftment was insufficient to rescue the mice from BM matrixnecrosis and complete depletion of hematopoietic elements (Table 1 andnot shown), the intestinal mucosa revealed multiple areas of activelyregenerating crypts. These data indicate that Bax deficiency mimics theprotection against GI lethality conferred by ASMase deficiency. Itshould be noted that Bax and Bak deficiencies did not impact the p53mediated epithelial apoptosis at crypt positions 4-5. Moreover, Bax andBak do not overlap functionally in the intestinal microvascular system.Additional support can be found in J. A. Rotolo, et al, Int. J.Radiation Oncology Biol. Phys., Vol. 70, No. 3, 804-815(2008),incorporated herein by reference.

Certain embodiments of the invention are directed to methods fortreating or preventing radiation damage or GI syndrome (and the otherenumerated diseases that are discussed below) in a subject byadministering an antisense nucleotide or siRNA that inhibits theendogenous expression of the target protein ASMase or Bak or Bax in thepatient. Other embodiments are directed to methods to treat or preventradiation disease or GI syndrome by administering imipramine in amountsthat ameliorate one or more symptoms of the disease. A therapeuticamount of antisense that inhibits ASMase for example, or imipramine canbe determined by routine experimentation as an amount that reducesASMase activity or expression in a biological sample from a human (ormammalian) subject compared to pretreatment levels.

The respective antisense nucleotide is one wherein at least a portion ofthe antisense nucleotide, typically 8-50 consecutive nucleotides) iscomplementary to and specifically hybridizes with the gene or mRNAencoding the target ASMase, Bax or Bak. The GenBank accession number forASMase is NP_(—)000534, incorporated herein as SEQ ID NO: 1. The GenBankaccession number for Bax is NP_(—)004315.1, incorporated herein as SEQID NO: 2. The GenBank accession number for Bak is NP_(—)001179.1,incorporated herein as SEQ ID NO: 3. As is described below, a person ofskill in the art can design a variety of antisense nucleotides andsiRNAs to disrupt either transcription or translation of the target geneor mRNA, respectively, to reduce expression of ASMase, Bax, or Bak.Antisense nucleic acids for use in this invention therapeutically totreat or prevent the enumerated diseases include cDNA, antisense DNA,antisense RNA, and small interfering RNA, that are sufficientlycomplementary to the target gene or mRNA encoding the target protein topermit specific hybridization to the respective gene or mRNA, therebyreducing expression of the target protein in the animal compared topretreatment levels.

In particular embodiments the enumerated diseases are treated orprevented by administering a therapeutic amount of an antisense nucleicacid from 8-50 nucleotides in length that specifically hybridizes to SEQID NO. 2 which is the cDNA sequence for human ASMase Accession No. NM000543; or SEQ ID NO. 4 which is the CDNA sequence for human BaxAccession No. NM 138761; or SEQ ID NO: 5 which is the cDNA sequence forhuman BAK Accession No. NM 001188. In another embodiment the antisenseis directed to various regions of the respective genomic DNAs that wouldblock transcription of the respective gene, ASMase SEQ ID NO. 7, Bax SEQID NO: 8, or Bak SEQ ID NO: 9. A patient could be treated with acombination of these antisense nucleic acids in a single preparation orin different preparations administered on the same or different days toreduce expression of ASMase (protein sequence SEQ ID NO. 1), or Bax(protein sequence SEQ ID NO.3); or Bak (protein sequence SEQ ID NO. 5).Alternatively, treatment could be achieved by administering theappropriate siRNA to reduce expression of one or more of the targetedproteins ASMase, Bax or Bak. More details regarding antisense technologyare set forth below.

For the purpose of this invention, a therapeutically effective amount ofa compound is an amount that achieves the desired biologic ortherapeutic effect, namely an amount that prevents, reduces orameliorates one or more symptoms of the enumerated diseases beingtreated or prevented. A starting point for determining an effectivetherapeutic amount of antisense or siRNA is an amount that reducesexpression of the targeted protein ASMase, or Bax or Bak in a biologicalsample taken from a subject. The therapeutic amount of imipramine can besimilarly determined.

Treatment and Prevention of IR-Induced Diseases and GI Syndrome withAnti-Ceramide Antibodies

We conducted in vitro studies in Jurkat T cells showing thatsequestration of ceramide with anti-ceramide antibody inhibitedceramide-mediated raft clustering, thereby attenuating apoptosis andimproving clonogenic survival (FIG. 4). To determine the in vivo effectsof anti-ceramide on radiation-induced apoptosis, 100 μg the commerciallysold mouse anti-ceramide antibody MID 15B4 or isotype control IgM wasadministered intravenously to C57BL/6 mice 30 min prior to 15 Gy TBI.Anti-ceramide infusion abrogated endothelial apoptosis 4 hrs post 15 Gy,decreasing the incidence of massive apoptosis (>10 apoptotic cells pervillus) from 56.9% in IgM treated controls to 13.7% (FIGS. 5B and D),pharmacologically recapitulating the protection afforded by geneticinactivation of ASMase (14.9%). These findings showed thatanti-ceramide-mediated protection of endothelium impacted GI stem celllethality, and thereby enhance overall animal survival. Antagonism ofendothelial apoptosis enhanced survival of crypt stem cell clonogens,evidenced by increased incidence of surviving crypts 3.5 days following15 Gy irradiation. As anti-ceramide attenuated endothelial apoptosis,pharmacologically recapitulating the asmase^(−/−) phenotype, we testedthe impact on crypt survival. Pretreatment of C57BL/6 mice withanti-ceramide prior to 15 Gy TBI resulted in a crypt surviving fractionof 1.3×10⁻¹, over 1 log of protection over the 9.3×10⁻³ survivingfraction exhibited by littermates treated with irrelevant IgM prior to15 Gy TBI (FIG. 5A). Irrelevant IgM antibody did not impact cryptsurvival compared with untreated C57BL/6 controls. Anti-ceramideantibody increased crypt survival to similar levels as geneticinhibition of ASMase (1.2×10⁻¹), demonstrating that pharmacologicinhibition of ceramide signaling mimics the protection afforded bygenetic inactivation of ASMase on crypt clonogen lethality in vivo. Itshould be noted that Bax and Bak deficiencies did not impact the p53mediated epithelial apoptosis at crypt positions 4-5. Moreover, Bax andBak do not overlap functionally in the intestinal microvascular system.Additional support can be found in J. A. Rotolo, et al, Int. J.Radiation Oncology Biol. Phys., Vol. 70, No. 3, 804-815(2008),incorporated herein by reference.

To assess whether anti-ceramide administration could recapitulate theasmase^(−/−) phenotype and increase animal survival following 15 Gy,C57BL/6 mice were pretreated with 50-100 μg anti-ceramide antibody orirrelevant IgM control and subjected to 15 Gy TBI. Within 16 hrs ofirradiation, mice were administered 3×10⁶ autologous bone marrow cellsintravenously. Consistent with previously published data, 15 Gy was 100%lethal in C57BL/6 control of IgM treated mice by day 7. Anti-ceramideantibody increased survival in a dose-dependent manner, with 100 μganti-ceramide pretreatment resulting in 60% survival 120 days followingirradiation (FIG. 5C). These findings correlate closely to the survivalof asmase^(−/−) mice administered autologous BMT following 15 Gy (F.Paris, Z. Fuks, A. Kang et al., Science 293 (5528), 293 (2001)).Autopsies revealed that mice receiving control IgM died with extensiveintestinal damage, including completely denuded crypts and villi, withonly partial damage to the bone marrow (not shown). These findings areconsistent with death from the GI syndrome. Conversely, autopsies ofmice that died following anti-ceramide pretreatment revealed typicalcharacteristics of bone marrow death, including extensive hemorrhage andnear complete depletion of hematopoietic elements (not shown). Thesemice exhibited intestinal mucosa in regenerative states, containinghyperplastic, chromophilic crypts covering most of the intestinalsurface. FIG. 5D shows micrographs of small intestine obtained 4 hrsfollowing 15 Gy-irradiation were stained by TUNEL. Apoptotic cells areindicated by brown-stained nuclei. Data (mean±SEM) were obtained fromminimum 150 villi from two independent experiments. These datademonstrate that anti-ceramide antibody effectively antagonizes ceramidesignaling in vivo, recapitulating the asmase^(−/−) phenotypepharmacologically and protecting against radiation-induced disease andGI syndrome

Based on this evidence for the role of extracellular ceramide inapoptosis, certain embodiments of the invention are directed to methodsfor treating or preventing radiation-induced disease and GI syndrome byadministering a therapeutic amount of one or more anti-ceramideantibodies or a biologically active fragment thereof, preferablyhumanized forms. These antibodies can be polyclonal or monoclonal. In apreferred embodiment the anti-ceramide antibody is monoclonal 2A2antibody or a biologically active fragment thereof, described below,preferably in humanized form. A new and effective monoclonalanti-ceramide antibody we discovered called 2A2 IgM is described indetail below. Previous work described above showed that statins(nystatin) also had beneficial effects in reducing apoptosis in in vitromodels. Therefore certain other embodiments include administering atherapeutic amount of an anti-ceramide antibody or biologically activefragment thereof, and one or more statins, administered alone or incombination to treat or prevent GI syndrome. The therapeutic agentsdescribed herein for combination therapy can be administered on the sameor on consecutive days.

Statins include any of a group of drugs that lower the amount ofcholesterol and certain fats in the blood. Statins inhibit a key enzymethat helps make cholesterol. The statins are divided into two groups:fermentation-derived and synthetic. The statins include, in alphabeticalorder (brand names vary in different countries):

Statin Brand name Derivation Atorvastatin Lipitor, Torvast SyntheticCerivastatin Lipobay, Baycol. (Withdrawn Synthetic from the market inAugust, 2001 due to risk of serious adverse effects) Fluvastatin Lescol,Lescol XL Synthetic Lovastatin Mevacor, Altocor Fermentation-derivedMevastatin — Naturally-occurring compound. Found in red yeast rice.Pitavastatin Livalo, Pitava Synthetic Pravastatin Pravachol, Selektine,Lipostat Fermentation-derived Rosuvastatin Crestor Synthetic SimvastatinZocor, Lipex Fermentation-derived. (Simvastatin is a synthetic derivateof a fermentation product) Simvastatin + Ezetimibe Vytorin Combinationtherapy Lovastatin + Niacin Advicor Combination therapy extended-releaseAtorvastatin + Amlodipine Caduet Combination therapy - BesylateCholesterol + Blood Pressure LDL-lowering potency varies between agents.Cerivastatin is the most potent, followed by (in order of decreasingpotency) rosuvastatin, atorvastatin, simvastatin, lovastatin,pravastatin,

2A2 Anti-Ceramide Monoclonal IgM Antibody

A flow chart of the strategy used to generate novel anti-ceramideantibodies with potent in vivo activity is shown in FIG. 6. In order tomake the antibody, we first needed to develop an antigen that wasimmunogenic enough t o generate a strong antibody response from aninoculated host. BSA-conjugated ceramide was generated by synthesizingBSA-conjugated C₁₆ fatty acid onto a sphingoid base. FIG. 7) inset.Validation of the Antigen for antibody screening was performed by ELISAassay, in which decreasing amounts of Antigen were fixed to a plate.After blocking each well, the plate was then incubated withanti-ceramide MID15B4 antibody (1:100) commercially available fromAxxora LLC, San Diego Calif. followed by horseradishperoxidase-conjugated anti-mouse IgM. OD was assessed followingadministration of HRP substrate at 650 nm. The BSA-ceramide ELISAidentified enhanced binding activity in supernatant #3673 followingimmunization with Kaposi sarcoma cells. FIG. 8. Binding of plasmasamples obtained from immunized mice by ELISA at 1:100 dilutionidentified higher binding of ceramide by sample #3673 vs. #3674. Bindingactivity remained following immortalization of antibody producing Bcells (sn73-I-C6), enabling the isolation of monoclonal 2A2 IgM withanti-ceramide binding activity (not shown). Karposi immunization wasintended to generate a strong immune response which would result ingeneration of a panel of antibody-producing B cells. Theantibody-containing supernatant from the hybridomas generated from theseB cells was then screened against the BSA-ceramide ELISA. Supernatantsthat tested positive in the assay were isolated, eventually resulting inpurification of clone 2A2. More details are set forth in Example 4.

Purified monoclonal 2A2 antibody was isolated from supernatant #3673.Elisa revealed that the 2A2 mouse monoclonal IgM bound to BSA-ceramide.FIG. 9. Elisa showed significantly more binding capacity of 2A2 vs.control IgM. We show that the 2A2 antibody works in vivo and we wereable to humanize it for clinical use. Methods for humanizing theantibody and others are set forth in Example 1.

Consistent with our earlier observations in vitro with commercialantibodies, purified 2A2 antibody antagonized radiation-inducedapoptosis in vitro. Preincubation of Jurkat cells with 2A2 monoclonalanti-ceramide antibody (25-100 micrograms/mL) inhibited 8 Gy-inducedapoptosis. FIG. 10; quantified as in FIG. 4B. Calculation of apoptosisinhibition was performed relative to the mean apoptosis of untreatedJurkat cells prior to 8 Gy. Other anti-ceramide antibodies 1H4, 15D9 and5H9, generated by immunization of mice with C₁₆ ceramide, can behumanized using techniques known in the art, and come within the scopeof this invention for treatment or prevention of GI syndrome, and alsoGvHD, autoimmune diseases and inflammation which are discussed below.

In the next series of experiments we showed that 2A2 enhanced cryptsurvival following 15 Gy in vivo. Pretreatment of C57BL/6 mice withincreasing doses of 2A2 anti-ceramide (0-750 micrograms) improved cryptsurvival at the critical 3.5 day time point following 15 Gy TBI. FIG.11(A). 2A2 anti-ceramide antibody increased crypt survival following8-15 Gy total body irradiation by a dose-modifying factor (DMF) of 1.2.FIG. 11(B). Crypt survival was determined as in FIG. 5C. In our animalexperiments we used 750 micrograms of antibody per 35 gram mouse. Thelocation of ceramide on the surface of activated T cells is particularlyimportant for treating or preventing any of the enumerated diseasesusing antibody therapy because this target protein is accessible to theantibodies.

In other experiments we discovered that anti 2A2 antibody also improvedsurvival of C57BL/6 mice exposed to 14-17 Gy single-dose radiation. FIG.12. C57BL/6 mice were irradiated with 14-17 Gy TBI with or without 750micrograms 2A2 15 min prior to IR. Mice were infused with 3×10⁶autologous bone marrow cells within 16 hour of IR. Survival wasmonitored and expressed via Kaplan-Meier parameters. Statisticalsignificance (P<0.05) was achieved at each dose.

Over a range of exposures, we found that 2A2 antibody attenuatedradiation-induced GI death in vivo, recapitulating the asmase^(−/−)phenotype. Necropsy results of mice sacrificed when moribund fromsurvival studies performed in FIG. 13. GI death was assessed whenproximal jejunum specimen appear >90% denuded of crypt-villi units andcrypt regeneration is absent. Bone marrow (BM) death was assessed whendecalcified femur sections reveal depletion of hematopoietic elementsand massive hemorrhage.

These results support embodiments of the present invention directed tothe 2A2 antibody itself, or fragment or variant thereof, preferably inhumanized form. In another preferred embodiment the human 2A2 antibody,or fragment thereof is administered in a therapeutically effectiveamount to treat or prevent radiation damage or GI syndrome. Whereantibody is used herein, it is also meant to include antibody fragmentsor variants as described below. Certain other embodiments are directedto a composition comprising an anti-ceramide antibody, preferablyhumanized, more preferably 2A2, and a statin in an amount that decreasescirculating cholesterol levels thereby increasing the efficacy of theanti-ceramide antibody. Another embodiment is directed to treating orpreventing radiation damage or GI syndrome by administering imipramine,and ASMase inhibitor presently used as an antipsychotic agent, eitheralone or in combination with anti-ceramide antibodies and/or statins.Another embodiment is a composition that includes the 2A2 antibody andimipramine or antisense or siRNA that targets ASMase or Bak.

Routine experimentation will determine the therapeutically effectiveamount of humanized monoclonal anti-ceramide antibody to use. The amountof anti-ceramide antibody to be administered therapeutically ranges fromabout lug to 100 ug/ml. This amount typically varies and can be anamount sufficient to achieve serum therapeutic agent levels typically ofbetween about 1 microgram per milliliter and about 10 micrograms permilliliter in the subject. In the context of the present invention,anti-ceramide antibodies are a type of neutralizing antibody thatprevents ceramide from blocking apoptosis.

As will be described below, we also discovered that 2A2 antibodyimproved survival after BMT by reducing GvHD and reduced the typicalcytokine storm seen with GvHD.

We discovered other monoclonal antibodies made in mice that wereimmunized with BSA-ceramide that when screened in a Jurkat cellapoptosis inhibition assay and showed protective effects that were dosedependent and comparable to 2A2. The isotypes of the three antibodieshave been established. 15D9 mAb is IgM, kappa. 1H4 and 5H9 mAbs aremIgG3, kappa. FIG. 25.

Certain embodiments of the invention are directed to these monoclonalantibodies 15D9, 1H4 and 5H9, and to fragments or variants thereof,preferably humanized for therapeutic use to treat radiation induceddiseases or GI syndrome, and as will be shown below also GvHD, otherautoimmune diseases and inflammation. Other embodiments are directed topharmaceutical compositions that include these monoclonal antibodies,fragments or variants, preferably in humanized form, and optional alosimipramine or statins or both.

One of the discoveries we made is that immunizing the host mice withKarposi Sarcoma cells as described in Example 4, generated effectiveanti-ceramide monoclonal antibodies with dramatic therapeutic effects asshown for example with the 2A2 antibody. We chose KS cells because forimmunization because they recapitulate activated endothelium which wehave shown is ceramide-rich. Because we immunized with activated cellsand not just with a pure ceramide antigen, the monoclonals we generatedmay cross react with other proteins besides ceramide. By cross react inthis application is meant that the monoclonals may react with otherproteins or protein complexes besides ceramide. For example the epitopeon ceramide that these monoclonals react with (i.e. have affinity for)may be shared with other cell surface proteins. Alternatively otherproteins may have a similar conformation at the antigenic site or theepitope may be part of a complex that displays the ceramide. Thereforecertain other embodiments of the invention are directed broadly tomonoclonal antibodies that cross-react with ceramide, wherein theantibodies are obtained by immunizing the host with whole cells.Humanized monoclonals including fragments are a preferred embodiment.The immunoglobulin subtype can be any subtype, IgG, IgM are preferredbut also IgA, IgE etc. may be effective.

Treatment and Prevention of Graft-vs.-Host Disease and AutoimmuneDisorders

The results presented below show for the first time thatASMase-generated ceramide is required for GVHD, therefore certainembodiments are directed to pharmacological methods to treat or preventGVHD by blocking ASMase (for example with imipramine or with antisensenucleic acids) or inactivate this cell surface ceramide (for examplewith anti-ceramide antibodies).

Autoimmune disorders are sustained adaptive immune responses directedagainst self antigens. T cells recognize self antigens as foreign due toincompatibilities between T cell and tissue major histocompatibilitycomplex (MHC) molecules and/or minor histocompatibilty antigens (mHAGs),cell surface molecules expressed on most cell types. Activated T cellsdirected against self antigens inflict tissue damage and chronicinflammation directly (via cytotoxic T cells, CTLs) or indirectly (viaantibody generation by T cell help to self-reactive B cells). Acutegraft-versus-host disease (GvHD), the primary complication ofhematopoietic stem cell transplantation, is a unique autoimmune-likedisorder arising from the differentiation and activation of alloreactivedonor T cells infused into an immunoablated host. In acute GvHD,recognition of alloantigens (major or minor mismatched) of the host bydonor T cells initiates an adaptive immune response including incipientdamage to host tissue and Type I cytokine (IFN-γ and IL-2) generation.This results in CTL clonal expansion and activation, that along with adeveloping macrophage-dependent “cytokine storm” comprised ofinflammatory cytokines (TNF-α and IL-1β) (D. A. Wall and K. C. Sheehan,Transplantation 57 (2), 273 (1994); G. R. Hill, W. Krenger, and J. L.Ferrara, Cytokines Cell Mol Ther 3 (4), 257 (1997); J. L. Ferrara, BoneMarrow Transplant 21 Suppl 3, S13 (1998), induces apoptosis in a selectset of target cells (A. C. Gilliam, D. Whitaker-Menezes, R. Korngold etal., J Invest Dermatol 107 (3), 377 (1996)), and consequent damage toassociated target organs (liver, intestines and skin)D. A. Wall, supra;G. F. Murphy, D. Whitaker, J. Sprent et al., Am J Pathol 138 (4), 983(1991)). Common symptoms of acute GvHD include severe weight loss,diarrhea, liver disease, rash and jaundice. FIG. 14 is a cartoonillustrating the immunopathology of GvHD.

High dose chemotherapy and radiation used in the treatment of many typesof leukemia and lymphomas additionally kill rapidly dividing bone marrowcells stem cells, resulting in immunoablation and necessitatingreconstitution of haematopoietic elements. Bone marrow transplantation(BMT) is a common and effective therapy for immune reconstitution inthese patients, as well as treatment for immune disorders includingaplastic anemia and severe combined immunodeficiency, in which infusionof bone marrow stem cells can correct deficiencies in red blood, Tand/or B cells. GvHD is the major complication associated with BMT,occurring in 30% of MHC matched, 50% of single MHC disparate and 70% ofdouble MHC disparate BMTs from related donors, and even higher when anon-related donor is used⁸⁶. Despite extensive research, there is nogood treatment. Acute GvHD typically develops within 3 months of BMT,during which specific organs including the gut, skin and liver aretargeted by cytotoxic T cells. Until now, the clinically relevantstrategies available to control GvHD are limited to T cell depletionfrom the allograft or non-specific immunosuppression aimed to controldonor T cell expansion and activation, approaches that increase thelikelihood of infection or neoplastic relapse in alreadyimmunocompromised patients.

Pharmacologic and genetic tools have enabled identification of severalkey mediators of CTL-induced apoptotic cell death at the cellular level.Liver, intestinal and skin apoptosis during acute GvHD is primarilymediated via CTL attack of host tissue via Fas-Fas ligand (FasL) (H.Kuwahara, Y. Tani, Y. Ogawa et al., Clin Immunol 99 (3), 340 (2001); C.Schmaltz, O. Alpdogan, K. J. Horndasch et al., Blood 97 (9), 2886(2001); K. Hattori, T. Hirano, H. Miyajima et al., Blood 91 (11), 4051(1998)) and TNF-TNFR pathways, with minimal contribution fromperforin/granzyme-mediated cell lysis. Inhibition of TNF-superfamilyreceptor signaling largely attenuated acute GvHD-associated mortality inmajor and minor mismatched models of murine allogeneic BMT, either bygenetic inactivation of donor T cell Fas ligand or TNF-α, orantibody-mediated neutralization of their cognate receptors CD95/Fas andTNFR, respectively. These studies established an essential role fordeath receptor signaling in donor CTL function in the pathogenesis ofacute GvHD, and suggested that promiscuous inhibition of TNF-superfamilysignaling might provide potent protection from GvH-associated pathology.

A number of recent studies identify a role for the sphingomyelin pathwayand its second messenger ceramide in autoimmune, liver and GItoxicities. Genetic inactivation of ASMase abrogates autoimmune mousemodels of phytohemagglutin (PHA)-induced Fas-dependent autoimmunehepatitis, during which induction of FasL on lymphocytes results inselective killing of hepatocytes expressing Fas, and activation of theHIV receptor gp120 by anti-CD4 antibody, in which depletion of CD4⁺ Tcells occurs due to upregulation of Fas and FasL on these cells.Further, evidence indicates that ceramide generation on the exoplasmicleaflet of the cell membrane occurs rapidly in the course ofischemia-reperfusion injury L. Llacuna, M. Mari, C. Garcia-Ruiz et al.,Hepatology 44 (3), 561 (2006)), and TNF-induced hepatocyte apoptosisthat leads to cirrhosis⁹³ and radiation-induced GI toxicity that hasalready been discussed using standardized animal models for thesedisease entities.

Acute GvHD, the primary complication of hematopoietic stem celltransplantation, is a well-defined autoimmune-like disorder mediated bydonor cytolytic T cell attack on host tissue. To evaluate thecontribution of target cell ASMase and ceramide in CTL-mediated tissueinjury and mortality during acute GvHD, a minorhistocompatability-incompatible allogeneic BM transplantation model ofLP/J donor (H-2^(b)) into C57BL/6 recipient (H-2^(b)) was selected.Lethally-irradiated C57BL/6 hosts of asmase^(+/+) or asmase^(−/−)background received 5×10⁶ T-cell depleted (TCD) LP/J BM cells, and GvHDwas induced by the addition of 3×10⁶ LP/J donor splenic T cells to theallograft. Transplantation of donor LP/J BM and T cells induced thedevelopment of GvHD in all recipients, albeit with reduced effect inasmase^(−/−) recipients as determined by Kaplan-Meier survival (FIG.15A) and clinical GvHD score (FIG. 15B) curves. GvHD survival wasincreased from 28.6% in asmase^(+/+) recipients of BM and T cells to84.6% in asmase^(−/−) recipients (p<0.005), and was accompanied byconsistently lower clinical scoring through day 90 in the asmase^(−/−)recipients (FIG. 15B and not shown), indicative of attenuated GvHD inasmase^(−l−) recipients. These results identify a role for ASMase inacute GvHD-induced mortality in a minor MHC-disparate allogeneic BMTmodel.

GvH-induced mortality is associated with injury to select organsincluding the ileum, liver and skin. Example 2 describes experimentsshowing that ASMase is required for GvHD target organ injury andapoptosis. Experiments in Example 2 show that ASMase deficiency largelyprotected GvHD-associated organ damage, decreasing scoring to 10.2±0.5and 7±0.1 in liver and small intestines, respectively (Table 1, p<0.005each for liver and intestine vs. asmase^(+/−) littermates). ASMasedeficiency also protected hosts from cutaneous keratinocyte apoptosisfollowing minor antigen-mismatched allogeneic BMT. FurtherGvHD-associated organ injury was shown to be associated with prominentintestine and skin apoptosis. These data identify a significantattenuation of GvHD-associated target organ damage and apoptosis,closely correlating with protection against GvHD morbidity andmortality, in asmase^(−/−) hosts compared to wild type littermatesacross both minor and major antigen disparities.

Additional experiments described in Example 3 show that ASMasedeficiency attenuated the cytokine storm associated with GvHD andinflammation generally, thus protecting the host from inflammation. Theexperiments showed that ASMase inactivation attenuated Th1/Th2 cytokineprofile and CD8 T cell proliferation in acute GvHD in recipients ofallogeneic BM and T cells. Our data showed deficient in vivo CD8⁺ CTLproliferation in asmase^(−/−) hosts, and biologically-relevantconsequences to the attenuation of serum proinflammatory cytokine levelsin these BMT recipients having GvHD. Thus, the in vivo data confirm arole for ASMase in GvH-associated morbidity, mortality and target organdamage. The impact of host ASMase deficiency affects GvHDpathophysiology primarily by to altering systemic factors (i.e.cytokines, number of circulating T cells) and not by inactivation of Tcell killing.

Administration of Anti-Ceramide Antibody Prevents GvHD

To assess whether ceramide-mediated raft clustering into membraneplatforms is an essential component of cytolytic T cell (CTL)-inducedapoptosis of hepatocytes, we blocked raft clustering pharmacologicallyand examined the impact on alloactivated CTL-induced hepatocyteapoptosis in vitro. To investigate whether ASMase deficiency renderstarget cells directly resistant to CTL-induced apoptosis, GvHD-mediatedtarget cell lysis was deconstructed in a 2-cell ex vivo model usingalloactivated splenic CTL effectors freshly-isolated from miceexhibiting active GvHD and naive C57BL/6 hepatocytes. Hepatocytes werechosen as target cells in this assay because they are a critical targetfor GvHD and utilize the sphingomyelin pathway for apoptosis. Under theconditions of our assay, 0.5 ×10⁶ hepatocytes were co-incubated with0-2×10⁶ alloactive splenic T cells isolated from LP/J BM+T cells→B6recipients. Co-incubation induced increased hepatocyte apoptosis from abaseline of 4.7±0.7% to 33.8±1.9% by 16 hr, as detected by nuclearmorphologic changes (FIGS. 16A). Consistent with data from a number ofin vivo studies, B6.MRL.lpr hepatocytes lacking a functional Fasreceptor were resistant to this mode of CTL-induced apoptosis (FIG. 16A,left panel), while selective inhibition of the granuleexocytosis-mediated cytolytic pathway by 2 hr incubation with 100 ng/mlCMA had no effect on hepatocyte apoptosis (FIG. 16A, right panel). Thesestudies indicate that CTL-mediated hepatocyte apoptosis in this modelrequires Fas but not perforin/granzyme signaling. Although prior studiesshowed that Fas levels were unaltered in asmase^(−/−) hepatocytes,asmase^(−/−) hepatocytes were nonetheless resistant to apoptosis in theex vivo GvHD model at all doses of allogeneic effector T cells from0.1-2×10⁶ (FIG. 16B), and at all times from 4-48 hr (data not shown).

Whereas Fas-mediated apoptotic death receptor activation requiresgeneration of ceramide-rich platforms in some cell systems, CTL-inducedplatform generation was assessed in asmase^(+/+) and asmase hepatocytesin the ex vivo model of GvHD. Alloactivated T cells induced rapidgeneration of ceramide-rich platforms on the surface of the targethepatocytes 10 min following co-incubation (FIG. 16C). Platformgeneration increased within one minute, peaked at 10 min, and persistedfor over 60 min (FIG. 16D). Fas, required for apoptosis in this system,concentrated within the ceramide-rich platform as determined by confocalmicroscopy. In contrast, asmase^(−/−) hepatocytes were completelyresistant to ceramide-rich platform formation (FIG. 16C and quantifiedin FIG. 16D) and Fas concentration therein, demonstrating thatCTL-induced platform generation in hepatocytes was ASMase-dependent.Concomitantly, CTLs induced a 1.5±0.1 fold overall increase in ceramidesignal as determined by mean fluorescence intensity per pixel inasmase^(+/+) hepatocytes (p<0.005 compared to unstimulated controls)which did not occur in asmase^(−/−) hepatocytes, accounting for thedifference in intensity of the ceramide staining in the upper vs. lowerpanels of FIG. 16C.

To demonstrate that hepatocyte apoptosis was specificallyceramide-dependent and not a consequence of ASMase deficiency, we addedsub-apoptotic concentrations of exogenous C₁₆-ceramide (up to 500 nM) toasmase^(−/−) hepatocytes in the ex vivo GvHD assay. Consistent withrestoration of platform generation in human asmase^(−/'1) lymphocytes bylow dose long chain natural ceramide, a sublethal dose of exogenousC₁₆-ceramide (200 nM) restored platform formation in CTL-treatedasmase^(−/−) hepatocytes to 46.3±1.3% of cells, demonstrating thatASMase-mediated ceramide generation drives platform formation.C₁₆-ceramide nearly completely restored CTL-induced apoptosis toasmase^(−/−) hepatocytes (FIG. 16E), bypassing the requirement fortarget cell ASMase. In contrast, C₁₆-dihydroceramide, thebiologically-inactive analog of C₁₆-ceramide, failed to restoreCTL-induced platform generation (not shown) or hepatocyte apoptosis(FIG. 16E). These data demonstrate unequivocally that CTL-inducedhepatocyte apoptosis ex vivo requires target cell ceramide generationfor efficient cell death induction, consistent with in vivo protectionfrom acute GvHD observed in asmase^(−/−) mice. Furthermore,pharmacologic disruption of cell surface rafts, sites of sphingomyelinconcentration, with the cholesterol-chelating agent nystatin abrogatedCTL-induced ceramide-rich platform generation (not shown) and completelyinhibited 2×10⁶alloactivated CTL-induced hepatocyte apoptosis (FIG.16F). Exogenous C₁₆-ceramide was unable to overcome cholesterolchelation (FIG. 16F), suggesting that ceramide-rich platform formationrequires functional rafts for signaling, possibly serving as a site forpre-assembly of the apoptotic machinery.

To show the generic role of target cell ASMase in T cell-induced lysis,we used two standardized assays for T cell function; MLR-primedCTL-induced splenocyte lysis (FIG. 17) and in vitro activation-inducedcell death (AICD) of T cells (FIG. 18). The role of target cell ASMasewas assessed in an in vitro activated MLR, in which C57BL/6 splenocytetarget cells of asmase^(+/+) or asmase^(−/−) genotype were stimulatedfor 48 hrs with mitogen, washed and chromium labeled. These cells werethen coincubated with effector Balb/c splenocytes activated for 5 daysby culture with irradiated C57BL/6 splenocytes. Chromium release wasquantified and the % lysis was calculated. To test the ability of invitro activated splenocytes to lyse asmase^(−/−) target cells, Balb/ceffector T cells were activated by culture for 5 days with 5×10⁶irradiated (2 Gy) C57BL/6 splenocytes. Target asmase^(+/+) orasmase^(−/−) C57BL/6 splenocytes were prepared by stimulation with 10mg/ml conA for 48 hrs and ⁵¹Cr labeled to assess CTL-mediated chromiumrelease. Washed splenocytes cells were coincubated with increasingratios of activated effector Balb/c T cells, and ⁵¹Cr release wasquantified 6 hour thereafter. FIG. 19A.

To evaluate a potential generic role for target cell ASMase in platformgeneration in T cell-induced lysis, we tested two standardized assaysfor T cell-mediated cytolysis, MLR-primed CTL-induced splenocyte lysis,and in vitro AICD of T cells. For the in vitro activated splenocytelysis assay, BALB/c effector T cells were stimulated by culture for 5days with 5×10⁶ irradiated (2 Gy) C57BL/6 splenocytes, and thereaftercoincubated with asmase^(+/+) or asmase^(−/−) target C57BL/6 splenocytesprepared by stimulation with 10 □g/ml conA for 48 hr. Co-incubation ofeffector T cells with asmase^(+/+) target splenocytes, MitotrackerRed-labeled for detection, resulted in rapid generation on target cellsof ceramide-rich platforms. Within 5 min of co-incubation, platformincidence on target asmase^(+/+) splenocytes increased from 4.7±2.1 to25.8±6.6% (p<0.01; FIG. 19A). Fas colocalized within these ceramide-richplatforms (data not shown). Platform generation was absent inasmase^(−/−) splenocytes, apparent in only 5.9±3.5% of the population 10min post stimulation. Furthermore, MLR-primed CTLs induced 43.2±6.5%asmase^(+/+) splenocyte lysis at an effector:target ratio of 50:1 (FIG.19B), attenuated to 7.3±2.5% lysis in asmase^(−/−) target cells, asquantified by ⁵¹Cr-release assay. Sublethal exogenous 500 nMC₁₆-ceramide restored both platform generation and CTL-induced lysis ofasmase^(−/−) target splenocytes, whereas C₁₆-dihydroceramide wasineffective in both events (not shown and FIG. 19C). These data indicatethat in vitro-primed CTL lysis of conA blasts activates target cellplatform generation in an ASMase-dependent manner, and that platformsare required for efficient lysis of target cells in these models.

To define a role for target cell ASMase in AICD, asmase^(+/+),asmase^(−/−) or MRL.lpr (FasR^(−/−)) T cells were stimulated for 24 hrwith 10 microgam/ml ConA, washed, rested in medium containing 20 U/mlrIL-2 for 24 hr, and finally AICD was initiated by restimulation inmedium containing 20 U/ml rIL-2 and increasing concentrations of platebound anti-CD3 mAb for 24 hr. AICD induction by anti-CD3 mAb conferredceramide-rich platform generation 4 hr following stimulation (FIG. 19D),evident in 26.4±1.6% of the population of asmase^(+/+) T cells comparedto 5.1±1.0% of asmase^(−/−) T cells (p<0.005). Confocal analysisidentified colocalization of the sphingolipid GM₁ (FIG. 19E) and Faswithin ceramide-rich platforms in asmase^(+/+) T cells.

To characterize the impact of platform generation on AICD, asmase^(+/+)and asmase^(−/−) T cells were induced to undergo AICD and apoptosis wasquantified 16 hr thereafter. Apoptosis in asmase^(+/+) T cells wasanti-CD3 mAb dose-dependent, reaching 32.9±5.2% apoptosis 16 hr postinduction (FIG. 19F). In contrast, apoptosis was abrogated in MRL.lpr(FasR^(−/−)) T cells (FIG. 19F), confirming the requirement for Fas/FasLinteraction in this system. While asmase^(−/−) T cells were induced toproliferate by anti-CD3 mAb equally well as asmase^(+/+) T cells, andwere equally effective as asmase^(+/+) T cells in lysis of tumor cells,they displayed near complete resistance to AICD-induced apoptosis invitro. asmase^(−/−) splenocytes failed to undergo detectable apoptosisabove background (8.8%) in response to up to 10 ng/ml anti-CD3 (p<0.05vs. asmase^(+/+) at 10 ng/ml anti-CD3; FIG. 19F). This occurred despitenormal upregulation of Fas and FasL (not shown). Natural long chainC₁₆-ceramide, but not C₁₆-dihydoceramide, completely restored platformgeneration in (not shown) and apoptosis of asmase^(−/−) T cellsstimulated to undergo AICD (FIG. 19G). These data provide compellingevidence that target cell ASMase-mediated ceramide-rich platformformation is required for T cell-induced apoptosis in bothantigen-disparate and fratricidic settings.

Treatment and Prevention of GvHD with Anti-Ceramide Antibody

We next looked at the effect of treatment with 2A2 anti-ceramideantibody IgM on survival following allogeneic bone marrow and T celltransplantation (FIG. 20). Using the same of LP/J donor (H-2^(b)) intoC57BL/6 recipient (H-2^(b)) model described in FIG. 15, we demonstratethat 2A2 antibody treatment significantly reduced objective indicia ofacute graft-versus-host disease in vivo, partially recapitulating theasmase⁴ phenotype. C57BL/6^(asmase+/+) or C57BL/6^(asmase−/−) werelethally irradiated with a split dose of 1100 cGy TBI. TheC57BL/6^(asmase+/+) group receiving 2A2 antibody received 750 microgramsantibody 15 min prior to the first half of split-dose TBI. Thereafter,all mice received intravenous injection of minor antigen-mismatched LPTCD-BM cells (5×10⁶) with splenic T cells (3×10⁶). Survival wasmonitored daily, and results were expressed by Kaplan-Meier survivalanalysis. Administration of 2A2 increased animal survival 100 daysfollowing transplantation from 12.5% to 60% (FIG. 20).

Given the role that ASMase plays in GvHD cytokine storm generation, wenext studied the ability of 2A2 Antibody to attenuate this adverseresponse. Serum was harvested from wild type, asmase^(−/−) and2A2-treated animals on day 7 following BMT from the group analyzed asdescribed above (FIG. 21). Serum interferon-gamma was quantified byELISA, according to manufacturer's protocol (R&D Systems). Our resultsshow that WT mice treated with 2A2 mAb responded similarly to ASMasedeficient mice, with an approximate 25% reduction in serum interferongamma.

These results show that GvHD and other T cell-mediated autoimmunediseases associated with an increase in pro-inflammatory cytokines canbe treated by administering a therapeutic amount of an anti-ceramideantibody, preferably 2A2 anti-ceramide monoclonal IgM or a biologicallyactive fragment thereof as is described herein. Our in vitro resultsshowed that nystatin also decreased T cell cytolysis ex vivo supportcertain embodiments wherein GvHD and autoimmune diseases are treated orprevented by administering one or more statins, preferably together witha humanized anti-ceramide antibody such as 2A2. Other embodiments aredirected to new compositions of one or more statins and one or moreanti-ceramide antibodies. As for treating GI syndrome, routineexperimentation will determine the effective amount of humanizedmonoclonal anti-ceramide antibody to use. The amount of anti-ceramideantibody to be administered therapeutically ranges from about 1 ug to100 ug /ml. This amount typically varies and can be an amount sufficientto achieve serum therapeutic agent levels typically of between about 1microgram per milliliter and about 10 micrograms per milliliter in thesubject.

The present studies define formation of ceramide-rich membrane platformsin the exoplasmic leaflet of the plasma membrane of target cells as acritical step in both microvascular apoptosis of the GI followingionizing radiation and lytic attack by CTLs upon immune targets. Thesestructures, which form within seconds of stimulation, are readilydetected by confocal or conventional microscopy as they are quite large,often reaching three microns in diameter. We have shown that genetic orpharmacologic disruption of platforms, for example by blocking ASMase orBax and by administering anti-ceramide antibodies, preventsradiation-induced cell death in vitro and in vivo, as well asCTL-mediated death in three distinct cellular models of CTL-induced cellkilling. Treatment with monoclonal anti-ceramide antibodies markedlyreduced multiple aspects of the pathophysiologic response ingastrointestinal radiation toxicity and standardized major and minormismatch models of GvHD.

Summary

The present studies provide new evidence that linkage betweenmicrovascular endothelial apoptosis and stem cell clonogen lethality isa generic mechanism for induction of radiation damage to the mouseintestines. Bax deficient mice provide evidence that it is theendothelial apoptotic response, rather than another event regulated byASMase signaling, that impacts the fate of irradiated crypt stem cellclonogens. The availability of a pharmacologic that can inhibit ASMase(such as imipramine, antisense nucleic acids or siRNA) is a valuabletherapeutic in patients requiring high dose abdominal radiation, withthe potential of selectively antagonizing GI toxicity without impactingtumor cell kill. Another valuable therapy is the use ofceramide-targeting pharmacologics such as anti-ceramide antibody toantagonize raft clustering and apoptosis of the GI early in the responseto accidental radiation exposure or large-scale radiological incident.Lethal GI toxicity following radiation exposure, for which there is nocurrent therapeutic option, is a primary concern of the NationalInstitute of Health in lieu of the current state of world affairs.

As the results we presented here show, ceramide-rich platforms arerequired for T cell-mediated autoimmune syndromes in vivo. ASMase hadbeen shown to regulate hepatocyte apoptosis in phytohemagglutin(PHA)-induced hepatitis, a model for acute T-cell mediatedautoaggressive liver disorder(S. Kirschnek, F. Paris, M. Weller et al.,J Biol Chem 275 (35), 27316 (2000)). PHA stimulates FasL induction onlymphocytes and upon their migration to the liver, hepatocytes arekilled by apoptosis, prompting hepatitis (K. Seino, N. Kayagaki, K.Takeda et al., Gastroenterology 113 (4), 1315 (1997)). ASMase deficiencyprotected hepatocytes from apoptosis despite normal upregulation oflymphocyte FasL. A role for ASMase in CD4-activated T cell-inducedcytolysis has been reported (Z. Q. Wang, A. Dudhane, T. Orlikowsky etal., Eur J Immunol 24 (7), 1549 (1994)). CD4 activation by the HIVreceptor molecule gp120, or by an agonistic anti-CD4 antibody, activatesthe Fas/FasL system and initiates apoptosis, whereas CD4⁺ T cellsdeficient in ASMase were unable to undergo apoptosis upon in vivoinjection of anti-CD4 antibody (S. Kirschnek, F. Paris, M. Weller etal., J Biol Chem 275 (35), 27316 (2000)). We have now shown thatASMase-generated ceramide is universally required in cytokine-mediated Tcell-induced apoptosis, and in preventing apoptosis of endothelialmicrovasculature which underlies the pathology of GI syndrome). We haveshown that blocking ASMase-generated ceramide with anti-ceramideantibodies also decreased serum proinflammatory cytokine expression.Thus any disease working through TNF superfamily receptor signalingincluding Fas/FasL and TNF/TNFR interactions can be treated or preventedby administering a therapeutic amount of an antibody that binds to theASMase-generated ceramide or by inhibition of ASMase or Bax.

The experiments described herein show for the first time that activationof host ASMase and formation of ceramide-rich platforms are criticalevents in instigation of acute GvHD. We showed this by the markedreduction in Type I cytokine production and CTL clonal expansion, stepsnecessary to feed forward GvHD, when mismatched alloactivated CTLs areadded to marrow transplants into asmase^(−/−) mice. As with GI syndrome,although different etiologies, GvHD responded to treatment withanti-ceramide antibodies and blocking ASMase or Bax expression.

The benefit of inactivation of ceramide-rich platforms is not restrictedto Fas-mediated biologic effects because other inflammatory cytokinesincluding but not limited to TNF, IL-1 and TRAIL similarly use ASMasefor transmembrane signal transmission. Hence, ASMase-generatedceramide-rich platforms represent a promiscuous target for GvHD andother immune disorders that utilize multiple cytokines to instigatepathophysiology.

Antibodies

An “antibody” refers to an intact immunoglobulin or to anantigen-binding portion thereof that competes with the intact antibodyfor specific binding. Antigen-binding portions may be produced byrecombinant DNA techniques or by enzymatic or chemical cleavage ofintact antibodies. Antigen-binding portions include, inter alia, Fab,Fab′, F(ab′).sub.2, Fv, dAb, and complementarity determining region(CDR) fragments, single-chain antibodies (scFv), chimeric antibodies,diabodies and polypeptides that contain at least a portion of animmunoglobulin that is sufficient to confer specific antigen binding tothe polypeptide.

An “immunoglobulin” is a tetrameric molecule. In a naturally-occurringimmunoglobulin, each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kDa) and one“heavy” chain (about 50-70 kDa). The amino-terminal portion of eachchain includes a variable region of about 100 to 110 or more amino acidsprimarily responsible for antigen recognition. The carboxy-terminalportion of each chain defines a constant region primarily responsiblefor effector function. Human light chains are classified as .kappa. and.lamda. light chains. Heavy chains are classified as .mu., .DELTA.,.gamma., .alpha., or .epsilon., and define the antibody's isotype asIgM, IgD, IgG, IgA, and IgE, respectively. Within light and heavychains, the variable and constant regions are joined by a “J” region ofabout 12 or more amino acids, with the heavy chain also including a “D”region of about 10 more amino acids. See generally, FundamentalImmunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989))(incorporated by reference in its entirety for all purposes). Thevariable regions of each light/heavy chain pair form the antibodybinding site such that an intact immunoglobulin has two binding sites.Immunoglobulin chains exhibit the same general structure of relativelyconserved framework regions (FR) joined by three hypervariable regions,also called complementarity determining regions or CDRs. The CDRs fromthe two chains of each pair are aligned by the framework regions,enabling binding to a specific epitope. From N-terminus to C-terminus,both light and heavy chains comprise the domains FR1, CDR1, FR2, CDR2,FR3, CDR3 and FR4. The assignment of amino acids to each domain is inaccordance with the definitions of Kabat Sequences of Proteins ofImmunological Interest (National Institutes of Health, Bethesda, Md.(1987 and 1991)), or Chothia & Lesk J. Mol. Biol. 196:901 917 (1987);Chothia et al. Nature 342:878 883 (1989).

An Fab fragment is a monovalent fragment consisting of the VL, VH, CLand CH I domains; a F(ab′).sub.2 fragment is a bivalent fragmentcomprising two Fab fragments linked by a disulfide bridge at the hingeregion; a Fd fragment consists of the VH and CH1 domains; an Fv fragmentconsists of the VL and VH domains of a single arm of an antibody; and adAb fragment (Ward et al., Nature 341:544 546, 1989) consists of a VHdomain. A single-chain antibody (scFv) is an antibody in which a VL andVH regions are paired to form a monovalent molecules via a syntheticlinker that enables them to be made as a single protein chain (Bird etal., Science 242:423 426, 1988 and Huston et al., Proc. Natl. Acad. Sci.USA 85:5879 5883, 1988). Diabodies are bivalent, bispecific antibodiesin which VH and VL domains are expressed on a single polypeptide chain,but using a linker that is too short to allow for pairing between thetwo domains on the same chain, thereby forcing the domains to pair withcomplementary domains of another chain and creating two antigen bindingsites (see e.g., Holliger, P., et al., Proc. Natl. Acad. Sci. USA90:6444 6448, 1993, and Poljak, R. J., et al., Structure 2:1121 1123,1994). One or more CDRs may be incorporated into a molecule eithercovalently or noncovalently to make it an immunoadhesin. Animmunoadhesin may incorporate the CDR(s) as part of a larger polypeptidechain, may covalently link the CDR(s) to another polypeptide chain, ormay incorporate the CDR(s) noncovalently. The CDRs permit theimmunoadhesin to specifically bind to a particular antigen of interest.

An antibody may have one or more binding sites. If there is more thanone binding site, the binding sites may be identical to one another ormay be different. For instance, a naturally-occurring immunoglobulin hastwo identical binding sites, a single-chain antibody or Fab fragment hasone binding site, while a “bispecific” or “bifunctional” antibody hastwo different binding sites.

An “isolated antibody” is an antibody that (1) is not associated withnaturally-associated components, including other naturally-associatedantibodies, that accompany it in its native state, (2) is free of otherproteins from the same species, (3) is expressed by a cell from adifferent species, or (4) does not occur in nature.

The term “human antibody” includes all antibodies that have one or morevariable and constant regions derived from human immunoglobulinsequences. In a preferred embodiment, all of the variable and constantdomains are derived from human immunoglobulin sequences (a fully humanantibody). These antibodies may be prepared in a variety of ways, asdescribed below.

A humanized antibody is an antibody that is derived from a non-humanspecies, in which certain amino acids in the framework and constantdomains of the heavy and light chains have been mutated so as to avoidor abrogate an immune response in humans. Alternatively, a humanizedantibody may be produced by fusing the constant domains from a humanantibody to the variable domains of a non-human species. Examples of howto make humanized antibodies may be found in U.S. Pat. Nos. 6,054,297,5,886,152 and 5,877,293, incorporated herein by reference.

The term “chimeric antibody” refers to an antibody that contains one ormore regions from one antibody and one or more regions from one or moreother antibodies.

Fragments or analogs of antibodies can be readily prepared by those ofordinary skill in the art following the teachings of this specification.Preferred amino- and carboxy-termini of fragments or analogs occur nearboundaries of functional domains. Structural and functional domains canbe identified by comparison of the nucleotide and/or amino acid sequencedata to public or proprietary sequence databases. Preferably,computerized comparison methods are used to identify sequence motifs orpredicted protein conformation domains that occur in other proteins ofknown structure and/or function. Methods to identify protein sequencesthat fold into a known three-dimensional structure are known. Bowie etal. Science 253:164 (1991).

Small Interfering RNA

Small interfering RNA can also be used therapeutically to treat orprevent GVHD, radiation induced diseases including GI syndrome,inflammation and autoimmune diseases in a subject by administering an siRNA at least a portion of which is complementary to and specificallyhybridizes with the mRNA encoding ASMase or Bax, in order to reduce orinhibit the respective expression, thereby blocking apoptosis.

US Patent Application 20040023390 (incorporated herein by reference)teaches that double-stranded RNA (dsRNA) can induce sequence-specificposttranscriptional gene silencing in many organisms by a process knownas RNA interference (RNAi). However, in mammalian cells, dsRNA that is30 base pairs or longer can induce sequence-nonspecific responses thattrigger a shut-down of protein synthesis and even cell death throughapoptosis. Recent work shows that RNA fragments are thesequence-specific mediators of RNAi (Elbashir et al., 2001).Interference of gene expression by these small interfering RNA (siRNA)is now recognized as a naturally occurring strategy for silencing genesin C. elegans, Drosophila, plants, and in mouse embryonic stem cells,oocytes and early embryos (Cogoni et al., 1994; Baulcombe, 1996;Kennerdell, 1998; Timmons, 1998; Waterhouse et al., 1998; Wianny andZernicka-Goetz, 2000; Yang et al., 2001; Svoboda et al., 2000).

In mammalian cell culture, a siRNA-mediated reduction in gene expressionhas been accomplished by transfecting cells with synthetic RNA nucleicacids (Caplan et al., 2001; Elbashir et al., 2001). The 20040023390application, the entire contents of which are hereby incorporated byreference as if fully set forth herein, provides methods using a viralvector containing an expression cassette containing a pol II promoteroperably-linked to a nucleic acid sequence encoding a small interferingRNA molecule (siRNA) targeted against a gene of interest.

As used herein RNAi is the process of RNA interference. A typical mRNAproduces approximately 5,000 copies of a protein. RNAi is a process thatinterferes with or significantly reduces the number of protein copiesmade by an mRNA. For example, a double-stranded short interfering RNA(siRNA) molecule is engineered to complement and match theprotein-encoding nucleotide sequence of the target mRNA to be interferedwith. Following intracellular delivery, the siRNA molecule associateswith an RNA-induced silencing complex (RISC). The siRNA-associated RISCbinds the target mRNA (such as mRNA encoding ASMase or Bax) through abase-pairing interaction and degrades it. The RISC remains capable ofdegrading additional copies of the targeted mRNA. Other forms of RNA canbe used such as short hairpin RNA and longer RNA molecules. Longermolecules cause cell death, for example by instigating apoptosis andinducing an interferon response. Cell death was the major hurdle toachieving RNAi in mammals because dsRNAs longer than 30 nucleotidesactivated defense mechanisms that resulted in non-specific degradationof RNA transcripts and a general shutdown of the host cell. Using fromabout 20 to about 29 nucleotide siRNAs to mediate gene-specificsuppression in mammalian cells has apparently overcome this obstacle.These siRNAs are long enough to cause gene suppression but not of alength that induces an interferon response.

The invention has been described in the foregoing specification withreference to specific embodiments. It will however be evident thatvarious modifications and changes may be made to the embodiments withoutdeparting from the broader spirit and scope of the invention. Thespecification and drawings are to be regarded in an illustrative ratherthan a restrictive sense.

Antisense Nucleic Acids

As used herein, the term “nucleic acid” refers to both RNA and DNA,including cDNA, genomic DNA, and synthetic (e.g., chemicallysynthesized) DNA. The nucleic acid can be double-stranded orsingle-stranded (i.e., a sense or an antisense single strand). As usedherein, “isolated nucleic acid” refers to a nucleic acid that isseparated from other nucleic acid molecules that are present in amammalian genome, including nucleic acids that normally flank one orboth sides of the nucleic acid in a mammalian genome (e.g., nucleicacids that flank an ARPKD gene). The term “isolated” as used herein withrespect to nucleic acids also includes any non-naturally-occurringnucleic acid sequence, since such non-naturally-occurring sequences arenot found in nature and do not have immediately contiguous sequences ina naturally-occurring genome.

An isolated nucleic acid can be, for example, a DNA molecule, providedone of the nucleic acid sequences normally found immediately flankingthat DNA molecule in a naturally-occurring genome is removed or absent.Thus, an isolated nucleic acid includes, without limitation, a DNAmolecule that exists as a separate molecule (e.g., a chemicallysynthesized nucleic acid, or a cDNA or genomic DNA fragment produced byPCR or restriction endonuclease treatment) independent of othersequences as well as DNA that is incorporated into a vector, anautonomously replicating plasmid, a virus (e.g., a retrovirus,lentivirus, adenovirus, or herpes virus), or into the genomic DNA of aprokaryote or eukaryote. In addition, an isolated nucleic acid caninclude an engineered nucleic acid such as a DNA molecule that is partof a hybrid or fusion nucleic acid. A nucleic acid existing amonghundreds to millions of other nucleic acids within, for example, cDNAlibraries or genomic libraries, or gel slices containing a genomic DNArestriction digest, is not to be considered an isolated nucleic acid.

Other embodiments of the present invention are directed to the use ofantisense nucleic acids (either DNA or RNA) or small interfering RNA toinhibit expression of ASMase or Bax or Bak or a biologically activefragment or variant thereof. The antisense nucleic acid can be antisenseRNA, antisense DNA or small interfering RNA. Based on the known sequenceof ASMase or Bax or Bak, antisense DNA or RNA that hybridizesufficiently to the respective gene or mRNA to turn off expression canbe readily designed and engineered using methods known in the art.

The isolated antisense or siRNA nucleic acid molecules for use in theinvention comprise a nucleic acid molecule which is a complement of thegene or mRNA sequence for the target: the gene (full genomic DNA)encoding ASMasae, identified as SEQ ID NO:1, the gene encoding humanBax, identified as SEQ ID NO: 2, or the gene encoding human Bak,identified as SEQ ID NO:3. A nucleic acid molecule which iscomplementary to a given nucleotide sequence is one which issufficiently complementary to the given nucleotide sequence that it canhybridize to the given nucleotide sequence thereby forming a stableduplex.

Antisense nucleic acid molecules, i.e., molecules which arecomplementary to a sense nucleic acid encoding a protein, e.g.,complementary to the coding strand of a double-stranded DNA molecule (orcDNA) or complementary to an mRNA sequence. Accordingly, an antisensenucleic acid can hydrogen bond to a sense nucleic acid. The antisensenucleic acid can be complementary to an entire ASMase, Bak or Bax codingstrand, or to only a portion thereof, e.g., all or part of the proteincoding region (or open reading frame). An antisense nucleic acidmolecule can be antisense to a noncoding region of the coding strand ofa nucleotide sequence encoding ASMase, Bak or Bax. The noncoding regions(“5′ and 3′ untranslated regions”) are the 5′ and 3′ sequences whichflank the coding region and are not translated into amino acids.

Given the coding strand sequences encoding ASMase (cDNA SEQ ID NO. 2,GENOMIC DNA SEQ ID NO. 7, Bax cDNA SEQ ID NO. 3, GENOMIC DNA SEQ ID NO.8, or Bak cDNA SEQ ID NO. 5, GENOMIC DNA SEQ ID NO. 9 disclosed herein,antisense nucleic acids of the invention can be designed according tothe rules of Watson and Crick base pairing. The antisense nucleic acidmolecule can be complementary to the entire coding region of ASMase, Bakor Bax mRNA, but more preferably is an oligonucleotide which isantisense to only a portion of the coding or noncoding region of ASMase,Bak or Bax mRNA. For example, the antisense oligonucleotide can becomplementary to the region surrounding the translation start site ofASMase, Bak or Bax. An antisense oligonucleotide can be, for example,about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length. Anantisense nucleic acid of the invention can be constructed usingchemical synthesis and enzymatic ligation reactions using proceduresknown in the art. For example, an antisense nucleic acid (e.g., anantisense oligonucleotide) can be chemically synthesized using naturallyoccurring nucleotides or variously modified nucleotides designed toincrease the biological stability of the molecules or to increase thephysical stability of the duplex formed between the antisense and sensenucleic acids, e.g., phosphorothioate derivatives and acridinesubstituted nucleotides can be used. Examples of modified nucleotideswhich can be used to generate the antisense nucleic acid include5-fluorouracil, 5-bromo uracil, 5-chlorouracil, 5-hodouracil,hypoxanthine, xanthine, 4-acetylcytosine,5-(carboxyhydroxylmethyl)uracil,5-carboxymethylaminomethyl-2-thouridine,5-carboxymethylaminometh-yluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-metnylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopenten-yladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thlouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-cxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)w,and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can beproduced biologically using an expression vector into which a nucleicacid has been subcloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest, described further inthe following subsection).

The antisense nucleic acid molecules of the invention are typicallyadministered to a subject or generated in situ such that they hybridizewith or bind to cellular mRNA and/or genomic DNA encoding the protein ofinterest to thereby inhibit expression of the protein, e.g., byinhibiting transcription and/or translation. The hybridization can be byconventional nucleotide complementary to form a stable duplex, or, forexample, in the case of an antisense nucleic acid molecule which bindsto DNA duplexes, through specific interactions in the major groove ofthe double helix. An example of a route of administration of antisensenucleic acid molecules of the invention include direct injection at atissue site. Alternatively, antisense nucleic acid molecules can bemodified to target selected cells and then administered systemically.For example, for systemic administration, antisense molecules can bemodified such that they specifically bind to receptors or antigensexpressed on a selected cell surface, e.g., by linking the antisensenucleic acid molecules to peptides or antibodies which bind to cellsurface receptors or antigens. The antisense nucleic acid molecules canalso be delivered to cells using the vectors described herein. Toachieve sufficient intracellular concentrations of the antisensemolecules, vector constructs in which the antisense nucleic acidmolecule is placed under the control of a strong pol II or pol IIIpromoter are preferred.

An antisense nucleic acid molecule of the invention can be analpha-anomeric nucleic acid molecule. An .alpha.-anomeric nucleic acidmolecule forms specific double-stranded hybrids with complementary RNAin which, contrary to the usual .beta.-units, the strands run parallelto each other (Gaultier et al. (1987) Nucleic Acids. Res. 15:6625-6641).The antisense nucleic acid molecule can also comprise a2′-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res.15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBSLett. 215:327-330). All of the methods described in the above articlesregarding antisense technology are incorporated herein by reference.

The invention also encompasses ribozymes. Ribozymes are catalytic RNAmolecules with ribonuclease activity which are capable of cleaving asingle-stranded nucleic acid, such as an mRNA, to which they have acomplementary region. Thus, ribozymes (e.g., hammerhead ribozymes(described in Haselhoff and Gerlach (1988) Nature 334:585-591)) can beused to catalytically cleave ASMase, Bak or Bax transcripts therebyinhibit translation of ASMase, Bak or Bax. A ribozyme having specificityfor an ASMase, Bak or Bax-encoding nucleic acid can be designed basedupon the nucleotide sequence of a ASMase, Bak or BaxcDNA disclosedherein. For example, a derivative of a Tetrahymena L-19 IVS RNA can beconstructed in which the nucleotide sequence of the active site iscomplementary to the nucleotide sequence to be cleaved in an ASMase, Bakor Bax-encoding mRNA. See, e.g., Cech et al. U.S. Pat. No. 4,987,071;and Cech et al. U.S. Pat. No. 5,116,742. Alternatively, ASMase, Bak orBax mRNA can be used to select a catalytic RNA having a specificribonuclease activity from a pool of RNA molecules. See, e.g., Barteland Szostak (1993) Science 261:1411-1418, incorporated herein byreference.\

As used herein, the term “nucleic acid” refers to both RNA and DNA,including cDNA, genomic DNA, and synthetic (e.g., chemicallysynthesized) DNA. The nucleic acid can be double-stranded orsingle-stranded (i.e., a sense or an antisense single strand). As usedherein, “isolated nucleic acid” refers to a nucleic acid that isseparated from other nucleic acid molecules that are present in amammalian genome, including nucleic acids that normally flank one orboth sides of the nucleic acid in a mammalian genome (e.g., nucleicacids that flank an ARPKD gene). The term “isolated” as used herein withrespect to nucleic acids also includes any non-naturally-occurringnucleic acid sequence, since such non-naturally-occurring sequences arenot found in nature and do not have immediately contiguous sequences ina naturally-occurring genome.

An isolated nucleic acid can be, for example, a DNA molecule, providedone of the nucleic acid sequences normally found immediately flankingthat DNA molecule in a naturally-occurring genome is removed or absent.Thus, an isolated nucleic acid includes, without limitation, a DNAmolecule that exists as a separate molecule (e.g., a chemicallysynthesized nucleic acid, or a cDNA or genomic DNA fragment produced byPCR or restriction endonuclease treatment) independent of othersequences as well as DNA that is incorporated into a vector, anautonomously replicating plasmid, a virus (e.g., a retrovirus,lentivirus, adenovirus, or herpes virus), or into the genomic DNA of aprokaryote or eukaryote. In addition, an isolated nucleic acid caninclude an engineered nucleic acid such as a DNA molecule that is partof a hybrid or fusion nucleic acid. A nucleic acid existing amonghundreds to millions of other nucleic acids within, for example, cDNAlibraries or genomic libraries, or gel slices containing a genomic DNArestriction digest, is not to be considered an isolated nucleic acid.

Pharmaceutical Compositions

The present invention also includes pharmaceutical compositions andformulations of the 2A2 humanized monoclonal antibody, optionallyformulated with one or more statins or imipramine. Other pharmaceuticalcompositions include the antisense nucleic acids and small interferingRNAs of the invention that are administered reduce ASMase or Bak or Baxexpression.

Pharmaceutical compositions of the present invention contain thetherapeutic agent (anti-ceramide antibody, enzyme inhibitors, antisensenucleic acids or si RNA) in an amount sufficient to prevent or treat theenumerated diseases: GVHD, radiation induced diseases including GIsyndrome, inflammation and autoimmune diseases in a subject. Thesepharmaceutical compositions are suitable for administration to a subjectin need of prophylaxis or therapy of GVHD, radiation induced diseasesincluding GI syndrome, inflammation and autoimmune diseases in asubject. The subject is preferably a human but can be non-human as well.A suitable subject can be an individual who is suspected of having, hasbeen diagnosed as having, or is at risk of developing one of theenumerated diseases. Therapeutic compositions may contain, for example,such normally employed additives as binders, fillers, carriers,preservatives, stabilizing agents, emulsifiers, buffers and excipientsas, for example, pharmaceutical grades of mannitol, lactose, starch,magnesium stearate, sodium saccharin, cellulose, magnesium carbonate,and the like. These compositions typically contain 1%-95% of activeingredient, preferably 2%-70% active ingredient.

Antibodies and antisense nucleotides and enzyme inhibitors or statins ofthe present invention can also be mixed with diluents or excipientswhich are compatible and physiologically tolerable. Suitable diluentsand excipients are, for example, water, saline, dextrose, glycerol, orthe like, and combinations thereof. In addition, if desired, thecompositions may contain minor amounts of auxiliary substances such aswetting or emulsifying agents, stabilizing or pH buffering agents.

In some embodiments, the therapeutic compositions of the presentinvention are prepared either as liquid solutions or suspensions, assprays, or in solid forms. Oral formulations usually include suchnormally employed additives such as binders, fillers, carriers,preservatives, stabilizing agents, emulsifiers, buffers and excipientsas, for example, pharmaceutical grades of mannitol, lactose, starch,magnesium stearate, sodium saccharin, cellulose, magnesium carbonate,and the like. These compositions take the form of solutions,suspensions, tablets, pills, capsules, sustained release formulations,or powders, and typically contain 1%-95% of active ingredient,preferably 2%-70%. One example of an oral composition useful fordelivering the therapeutic compositions of the present invention isdescribed in U.S. Pat. No. 5,643,602 (incorporated herein by reference).

Additional formulations which are suitable for other modes ofadministration, such as topical administration, include salves,tinctures, creams, lotions, transdermal patches, and suppositories. Forsalves and creams, traditional binders, carriers and excipients mayinclude, for example, polyalkylene glycols or triglycerides. One exampleof a topical delivery method is described in U.S. Pat. No. 5,834,016(incorporated herein by reference). Other liposomal delivery methods mayalso be employed (See, e.g., U.S. Pat. Nos. 5,851,548 and 5,711,964,both of which are herein incorporated by reference).

The formulations may also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.Such molecules are suitably present in combination in amounts that areeffective for the purpose intended. For example, the 2A2 antibody couldbe formulated with a statin or with imipramine.

Sustained-release preparations may also be prepared. Suitable examplesof sustained release preparations include semipermeable matrices ofsolid hydrophobic polymers containing the antibodies or fragments,nystatin, imipramine or combinations thereof, which matrices are in theform of shaped articles, e.g., films, or microcapsule. Examples ofsustained-release matrices include, but are not limited to, polyesters,hydrogels (for example, poly (2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides, copolymers of L-glutamic acid and yethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods.

The antibodies and antisense or siRNAs of the present invention may beadministered by any suitable means, including parenteral, subcutaneous,topical, intraperitoneal, intrapulmonary, and intranasal, and,intralesional administration (e.g. for local immunosuppressivetreatment). Parenteral infusions include intramuscular, intravenous,intraarterial, intraperitoneal, or subcutaneous administration. Inaddition suitable administration includes by pulse infusion,particularly with declining doses of the antibody. Preferably, thedosing is given by injections, most preferably intravenous orsubcutaneous injections, depending in part on whether the administrationis brief or chronic.

For the prevention or treatment of disease, the appropriate dosage ofantibody will depend on the type of disease to be treated, the severityand course of the disease, whether the drug is administered forpreventive or therapeutic purposes, previous therapy, the patient'sclinical history and response to the new drugs (2A2 antibody, etc.) andthe discretion of the attending physician. The antibodies andnucleotides or other drugs (imipramine and statins) are suitablyadministered to the patient at one time or over a series of treatments.

As mentioned above, the amount of anti-ceramide antibody to beadministered therapeutically ranges from about lug to 100 ug/ml. Thisamount typically varies and can be an amount sufficient to achieve serumtherapeutic agent levels typically of between about 1 microgram permilliliter and about 10 micrograms per milliliter in the subject. Thetherapeutic agents of the invention can be administered by one or moreseparate administrations, or by continuous infusion. For repeatedadministrations over several days or longer, depending on the condition,the treatment is sustained until the symptoms are sufficiently reducedor eliminated. The progress of this therapy is easily monitored byconventional techniques and assays, and may be used to adjust dosage toachieve a therapeutic effect.

A candidate dosage of antisense nucleic acid or siRNA for usetherapeutically can be determined initially by finding an amount thatreduces expression of the target protein in a biological sample from ahuman or animal.

In the foregoing specification, the invention has been described withreference to specific embodiments thereof. It will, however, be evidentthat various modifications and changes may be made thereto withoutdeparting from the broader spirit and scope of the invention. Thespecification and drawings are, accordingly, to be regarded in anillustrative rather than a restrictive sense.

EXAMPLES Example 1 Materials & Methods Cell Culture and Stimulation

Wild-type (clone E6-1), caspase 8^(−/−) (clone 19.2) and FADD^(−/−)(clone 12.1) Jurkat T lymphocytes were obtained from ATCC (Rockville,Md.). Cells were grown in a 5% CO₂ incubator at 37° C. in RPMI 1640medium supplemented with 10% heat-inactivated fetal bovine serum and 10mM Hepes (pH 7.4), 2 mM L-glutamine, 1 mM sodium pyruvate, 100 μMnonessential amino acids, 100 units/ml penicillin, and 100 μg/mlstreptomycin. Prior to stimulation with UV-C or anti-Fas, cells wereresuspended in fresh medium and allowed to acclimate for 4 hours. Jurkatcells were then treated with 50 ng/ml anti-FasCH-11 activating antibody(Upstate Biotechnology, Lake Placid N.Y.) or 50 Joules/m² UV-C using anFB-UVXL-1000 Crosslinker (Fisher Biotech, Pittsburgh Pa.), unlessotherwise indicated. For platform studies, Jurkat cells were incubatedwith CH-11 at 4° C. for 20 min to insure uniform receptor engagement,and warmed to 37° C. to initiate stimulation.

Where indicated, cells were pre-incubated with 10 μM z-VAD-fmk(Calbiochem, La Jolla Calif.), 30 μg/ml nystatin (Sigma-Aldrich,Milwaukee Wis.), 50 μM imipramine (Sigma-Aldrich) or 1 μg/ml mousemonoclonal anti-ceramide antibody MID15B4 (Alexis Biochemicals, SanDiego CA). Nystatin, imipramine and anti-ceramide studies were performedin RPMI containing 0.5% lipid-free fetal bovine serum (HyClone, LoganUtah). In each study, an aliquot of cells were stained with trypan blueto assess viability.

Mice and Bone Marrow Transplantation (BMT)

C3H/HeJ, C3HeB/FeJ, LP/J (“LP,” H-2^(b)), B10.BR (“B10,” H-2^(k)) andB6.MRL.lpr female mice, 8-12 weeks old, were purchased from JacksonLaboratories (Bar Harbor, Me.). SV129/C57BL/6^(asmase−/−) andC57BL/6^(Bax−/−) mice were inbred in our colony and genotyped aspreviously described [Horinouchi, 1995 #171; Knudson, 1995 #173].Backcrossing the asmase^(−/−) genotype onto the C57BL/6 background wasperformed by initially mating wildtype C57BL/6 females with maleSV129/C57BL/6^(asmase−/−) mice. Male F₁ mice with asmase^(+/−) genotypewere subsequently mated with C57BL/6 females. Thereafter, a matingprotocol of male asmase^(+/−) progeny with wildtype C57BL/6 female micewas continued for ten generations to obtain an asmase^(+/−) genotype inpure C57BL/6 background. Once backcrossing was established,C57BL/6^(asmase+/−) mice were interbred to obtain experimental animals.Male and female hosts were used in BMT experiments at 8-12 weeks of age.BMT protocols were approved by the Memorial Sloan-Kettering CancerCenter Institutional Animal Care and Use Committee. Mice were housed ina pathogen-free facility at Memorial Sloan-Kettering Cancer Center insterilized micro-isolator cages and received normal chow and autoclavedhyperchlorinated drinking water (pH 3.0). This facility is approved bythe American Association for Accreditation of Laboratory Animal Care andis maintained in accordance with the regulations and standards of theUnited States Department of Agriculture and the Department of Health andHuman Services, National Institutes of Health.

Bone marrow (BM) cells were removed aseptically from femurs and tibias.Donor marrow was T-cell depleted using anti-Thy-1.2 antibody andlow-TOX-M rabbit complement (Cedarlane Laboratories, Hornby, Canada).Splenic T cells were obtained by purification over a nylon wool column.Cells (5×10⁶ BM cells with or without splenic T cells) were resuspendedin Dulbecco's modified essential medium and transplanted by tail-veininfusion into lethally-irradiated recipients that had received 1100 cGytotal body irradiation (¹³⁷Cs source) as a split dose with 3 hrs betweendoses on day 0.

Cell culture medium for lymphocyte and bone marrow harvesting consistedof RPMI 1640 supplemented with 10% heat-inactivated fetal calf serum,100 U/mL penicillin, 100 μg/mL streptomycin, 2 mM L-glutamine, and 50 μM2-mercaptoethanol. Anti-murine CD16/CD32 Fc block (2.4G2),fluorochrome-labeled CD3 (145-2C11), CD4 (RM4-5), CD8 (53-6.7), CD62L(MEL-14), and Ly-9.1 (3007) antibodies were obtained from Pharmingen(San Diego, Calif.). Ammonium chloride red blood cell (RBC) lysisbuffer, concanavalin A (conA) and concanamycin A (CMA) were obtainedfrom Sigma (St. Louis, Mo.).

Ex Vivo Hepatocyte Culture

Hepatocytes were isolated by cannulation of the portal vein andretrograde in situ collagenase perfusion according to the methoddescribed by Klaunig⁹⁴. Briefly, livers were perfused with 20 mL Buffer1 (Krebs Ringer with glucose+0.1mM EGTA) followed by 25 mL Buffer 2[Krebs Ringer with glucose containing 0.2 mM CaCl₂ with 5,000 unitscollagenase type I (Sigma)] at a rate of 7 ml/min by peristaltic pump(Rainin Instrument LLC. Woburn, Mass.). Perfused livers were excised andminced in Buffer 2, filtered through a 100 μM cell strainer, washedtwice at 50×g and resuspended in RPMI 1640 complete medium containing10% fetal bovine serum. Viability was routinely greater than 90%.

Apoptosis Quantitation

Apoptosis was assessed in vitro by two different techniques. TUNELstaining was performed on cells permeabilized with 0.1% Triton X-100 and0.1% sodium citrate at 4° C. for 5 min, according to the manufacturer'sinstructions (Roche Molecular Biochemicals, Indianapolis Ind.).Alternately, stimulated cells were fixed with 2% paraformaldehyde,washed with phosphate buffered saline (PBS), and stained with 100 μl of24 μg/ml bis-benzimide trihydrochloride solution (Hoechst #33258;Sigma-Aldrich, Milwaukee Wis.) for 10 min. Morphologic changes ofnuclear apoptosis including chromatin condensation, segmentation andcompaction along the periphery of the nucleus or the appearance ofapoptotic bodies were quantified using an Axiovert S-100 Zeissfluorescence microscope as previously described ³³. A minimum of 200cells were examined per point.

Apoptosis was quantified in vivo in the endothelium of the laminapropria of the small intestines following TUNEL staining as described[Paris, 2001 #7]. Endothelial cells were identified by immunostainingusing an antibody against the endothelial cell surface marker CD31(PharMingen catalog no. 1951D) as previously published [Paris, 2001 #7].

Hepatocyte apoptosis was assessed on 0.5×10⁶ hepatocytes rested for 30min in complete medium and stimulated for 16 hrs at 37° C. in 5% CO₂with 1 μg/mL anti-Fas Jo2 antibody (Pharmingen) or 0-2×10⁶ splenic Tcells isolated from C57BL/6 mice 2-3 weeks following transplantation ofLP/J donor bone marrow and T cells as described. In some studies,hepatocytes were pretreated for 30 min with nystatin (50 μg/mL,Sigma-Aldrich, Milwaukee Wis.), washed in RPMI, and resuspended in RPMIsupplemented with 1% lipid-free FBS prior to stimulation.

Clonogenic Assay

Colony formation of Jurkat cells following UV-C or anti-Fas CH-11treatment was evaluated using a soft agar cloning assay as describedpreviously⁹⁵. Briefly, cells were preincubated with nystatin andanti-ceramide mAb, or vehicle and control IgM in RPMI+0.5% lipid-freefetal bovine serum, and stimulated with increasing doses of UV-C oranti-Fas for 4 hours. Subsequently, cells were suspended in RPMI mediumcontaining 20% FBS, 20 mM L-glutamine and 40% methylcellulose medium,and plated in triplicate. After 14-16 days of incubation, plates wereanalyzed by inverted microscope and aggregates with more than 20 cellswere scored as colonies. Colony formation for each condition calculatedin relation to values obtained for untreated control cells. Colonysurvival curves were calculated by least square regression analysis,using a modification of the FIT software program⁹⁶. The program fits thecurves by iteratively weighted least squares to each set ofdose-survival data, estimates the covariates of the survival curveparameters and the corresponding confidence regions, and plots thesurvival curve. It also derives curve parameters, such as the D_(o) (thereciprocal of the slope on the exponential portion of the curve,representing the level of radiosensitivity) and the N number (measuringthe size of the shoulder).

Platform Detection

Platforms were detected as previously described. Briefly, 1×10⁶ Jurkatcells were stimulated with UV-C or α-Fas, fixed with 2% paraformaldehydeat the indicated times, blocked in PBS containing 1% fetal bovine serum,and then washed with PBS. Cells were stained for the raft-localizedlipid GM1 with FITC-conjugated cholera toxin β-subunit (2 μg/ml;Sigma-Aldrich) for 45 mM at 4° C., washed twice in PBS containing 0.1%Triton X-100 and mounted in fluorescent mounting medium (Dako,Carpenteria Calif.). Fluorescence was detected using an Axiovert S-100Zeiss fluorescence microscope equipped with a SPOT digital camera. Thepercentage of cells containing platforms, i.e. those in which thefluorescence condenses onto less than 15% of the cell surface, wasdetermined by counting 150-250 cells per point. Alternately, platformswere identified using a mouse monoclonal anti-ceramide antibody MID 15B4IgM (1:50 dilution, Alexis Biochemicals), mouse monoclonal anti-FasCH-11 IgM (1:500 dilution, Upstate Biotechnology) or polyclonal rabbitanti-ASMase antibody 1598 (1:100 dilution) and detected usingCy3-conjugated anti-mouse or anti-rabbit IgM (1:500 dilution, RocheMolecular Biochemicals), respectively. Rabbit polyclonal anti-CD46(1:1000 dilution, Santa Cruz Biotechnology, Santa Cruz Calif.) was usedas a negative control. In some studies, confocal images were obtainedusing a Leica TCS SPZ upright confocal microscope. Alternatively,0.5×10⁶ hepatocytes were stimulated for the indicated times with CTLsand platform formation was assessed as described.

The rabbit polyclonal anti-ASMase antibody #1598 was generated againstfull-length FLAG-tagged human ASMase protein. Anti-sera was purifiedover a BIO-RAD T-Gel Column to obtain an IgG fraction that displaysspecific immunoreactivity by immunoblot assay at a concentration of 100ng.μl towards 100 ng of purified recombinant human ASMase or ASMase from25 μg of Jurkat cell lysates. At a concentration of 200 μg/μl, #1598quantitatively immunoprecipitates ASMase activity from 100 ng ofpurified ASMase and at a concentration of 200 ng/μl detects cell surfaceexpression of ASMase by flow cytometry or confocal immunofluorescencemicroscopy.

Diacylglycerol Kinase Assay (DGK)

Jurkat cells, stimulated with UV-C or CH-11, were incubated for theindicated times at 37° C. Stimulation was terminated by the addition of2 ml of chloroform:methanol:HCl (100:100:1,v/v/v), and ceramide wasquantified by the diacyglycerol kinase assay, as described⁹⁷.

Western Blot Analysis

Jurkat cells stimulated with UV-C or CH-11 were incubated for theindicated times at 37° C. Stimulation was terminated with ice-cold PBSand cells were lysed in RIPA buffer (25 mM HEPES (pH 7.4), 0.1% SDS,0.5% sodium deoxycholate, 1% Triton X-100, 100 mM NaCl, 10 mM NaF, 10 mMNa₂P₂O₇, 10 mM EDTA and 10 μg/ml each aprotonin and leupeptin). Sampleswere centrifuged at 14,000 g and the supernatants were added to 4×SDS-sample buffer. Lysates were separated on a 10% SDS-PAGE gel andtransferred onto nitrocellulose membranes. Caspase cleavage was detectedusing rabbit polyclonal antibodies against caspase 3 (BD PharMingen, SanDiego Calif.), caspase 8 (BD PharMingen) or caspase 9 (Cell SignalingTechnology, Beverly Mass.). Caspase 8 and FADD expression levels weredetected using mouse monoclonal anti-caspase 8 (clone 1-1-37; UpstateBiotechnology) or anti-FADD (BD PharMingen) antibodies, respectively.

Flow Cytometry Analyses

To detect surface ASMase by FACS, Jurkat cells were stimulated with 50J/m² UV-C or 50 ng/ml CH-11 at 37° C. Stimulation was terminated after 1mM, the time of maximal ASMase translocation, by addition of ice-coldwashing buffer (PBS containing 1% FCS and 0.1% NaN₃), and cells wereblocked on ice for 20 min using the same buffer supplemented withisotype control rabbit IgG (20 μg/ml). Cells were re-washed andincubated for 45 min with 1 μg/ml of polyclonal anti-ASMase 1598antibody in PBS, followed by washing and incubation with Cy3 conjugatedanti-rabbit IgG. 10,000 cells were analyzed using a FACScan flowcytometer (Becton Dickinson, Franklin Lakes N.J.).

Harvested splenocytes were washed, incubated with CD16/CD32 FcR block onice for 15 mM, subsequently incubated with primary antibodies for 45 mM,washed, resuspended in FACS buffer (PBS+2% BSA+0.1% NaN₃) and analyzedon a FACScan flow cytometer with CellQuest software (Becton Dickinson).For CFSE staining, RBC-lysed LP/J or B10.BR splenocytes were positivelyselected as per manufacturer's instruction with anti-CD3 microbeads(Miltenyi, Auburn, Calif.), stained in 2.5 μM carboxyfluoresceindiacetate succinimidyl ester (CFSE) and 15-20×10⁶ stained cells weretransplanted into allogeneic (B6) recipients of either asmase^(+/+) orasmase^(−/−) background. Splenocytes from these animals were harvested72 hrs thereafter, stained with fluorochrome-conjugated antibodies forsurface antigens and FACS analysis was carried out as above.

ASMase Activity Assay

ASMase activity was measured using a fluorescence-based,high-performance liquid chromatographic (HPLC) assay⁹⁸. Briefly, 5×10⁶Jurkat cells were stimulated with 50 J/m² UV-C or 50 ng/ml CH-11 at 37°C., and at the indicated times washed with ice-cold PBS and lysed on icein NP-40 buffer (150 mM NaCl, 25 mM Tris HCl pH 7.5, 10% glycerol, 1%NP-40, 2 mM EDTA, 0.1 M DTT supplemented with PMSF, leupeptin andprotease inhibitor cocktail). ASMase activity was measured by incubatingan equal volume of lysate in assay buffer [500 μM BODIPY-C₁₂sphingomyelin (Molecular Probes, Eugene Oreg.), 0.1 mM ZnCl₂, 0.1 Msodium acetate pH 5.0 and 0.6% Triton X-100] for 60 min at 37° C.Thereafter, the reaction was stopped by 10× dilution in ethanol and 5 μlof the assay mixture was sampled by a WIPS 712 (Waters Corp., MilfordMass.) auto-sampler equipped with a 20×4 mm reverse-phase Aquasil C₁₈column (Keystone Scientific, Bellefonte Pa.). The reaction product,BODIPY-C₁₂ ceramide, was specifically separated from substrate within0.4-0.5 min by isocratic elution with 95% Me0H at a flow rate of 1.2ml/min. Fluorescence was quantified using a Waters 474 (Waters Corp.)fluorescence detector set to excitation and emission wavelengths of 505and 540 nm, respectively. The amount of product generated was calculatedusing a regression equation derived from a standard curve establishedfor known amounts of BODIPY-C₁₂ ceramide standard. Alternatively, ASMaseactivity was quantified by radioenzymatic assay using[¹⁴C-methylcholine]sphingomyelin (Amersham Biosciences, Piscataway,N.J.) as substrate, as described ¹⁴. Briefly, Jurkat cells were lysed inPBS containing 0.2% Triton X-100 at the indicated times after 50 J/m²UV-C or 50 ng/ml CH-11 stimulation. Post nuclear supernatants wereassayed for activity in 250 mM sodium acetate, pH 5.0 supplemented with0.1 mM ZnCl₂, 1 mM EDTA and 0.1% Triton X-100 in the presence ofsubstrate. Reactions were terminated after 1 hour with CHCl₃:MeOH:1N HCl(100:100:1, v/v/v), and product was quantified by scintillation counter.As both assays yielded identical fold increases after UV-C or Fasstimulation, these data were collated. However, BODIPY-C₁₂ sphingomyelinwas less efficiently catalyzed resulting in a lower Vmax as determinedby Michaelis Menton kinetic analysis. Thus, baseline ASMase specificactivity, derived from the radioenzymatic assay, is displayed throughoutthe manuscript.

Radiation and Tissue Preparation

TBI was delivered with a Shepherd Mark-I unit (Model 68, SN643)operating ¹³⁷Cs sources. The dose rate was 2.12 Gy/min. To collect smallintestinal samples, mice were sacrificed by hypercapnia asphyxiation and2.5 cm segments of the proximal jejunum were obtained at 2 cm from theligament of Trietz. Tissue samples were fixed by overnight incubation in4% neutral buffered formaldehyde and embedded in paraffin blocks. Toevaluate intestinal tissue responses to radiation, transverse sectionsof the full jejunal circumference (5 μm thick) were obtained bymicrotomy from the paraffin blocks, adhered to polylysine-treated slidesand deparaffinized by heating at 90° C. for 10 minutes and at 60° C. for5 minutes, followed by two xylene washes for 5 minutes, and stained withhematoxylin and eosin according to a standard protocol. To determine thecauses of death after TBI, autopsies were performed within 60 min ofanimal death or when terminally-sick animals displaying an agonalbreathing pattern were sacrificed by hypercapnia asphyxiation. Tissuespecimens were collected from all animals, fixed in formaldehyde, andstained with hematoxylin.

Crypt Microcolony Survival Assay

The microcolony survival assay was performed as described by Withers andElkind⁹⁹. Briefly, 3.5 days after irradiation mice were sacrificed byhypercapnia asphyxiation and samples of the small intestine wereharvested and prepared for histological staining as described above.Surviving crypts were defined as containing 10 or more adjacentchromophilic non-Paneth cells, at least one Paneth cell and a lumen. Thecircumference of a transverse cross section of the intestine was used asa unit. The number of surviving crypts was counted in eachcircumference. 10-20 circumferences were scored per mouse and 2-4 micewere used to generate each data point. Data were reported as mean±SEM.

Assessment of GVHD

Survival was monitored daily, and ear-tagged animals in coded cages wereindividually scored weekly for 5 clinical parameters (weight loss,hunched posture, decreased activity, fur ruffling, and skin lesions) ona scale from 0 to 2. A clinical GVHD score was generated by summation ofthe 5 criteria scores (0-10) as described by Cooke et al¹⁰⁰. GVHD targetorgan pathology for bowel (terminal ileum and ascending colon), liverand skin (tongue and ear) was assessed by one individual (J.M.C. forliver and intestinal pathology, G.F.M. for cutaneous pathology) in ablinded fashion on 10% formalin-buffered phosphate-preserved,paraffin-embedded and hematoxylin/eosin-stained 5 μM histopathologysections with a semiquantitative scoring system. Briefly, bowel andliver were scored for 19 to 22 different parameters associated with GVHDas described¹⁰¹ and skin was evaluated for the number of dyskeratoticand apoptotic cells, as published¹⁰². Villus and crypt cell apoptosiswere scored on formalin-preserved, paraffin-embedded, TUNEL-stained andhematoxylin/eosin-counterstained sections, as described^(18,62).

ELISA

Enzyme-linked immunosorbent assays (ELISA) for serum IL-1β, IL-2, IFN-γand TNF-α levels was performed according to the manufacturer'sinstructions (R&D, Minneapolis, Minn.).

Activation-Induced Cell Death (AICD) Assay

Harvested splenocytes were enriched for T lymphocytes by nylon woolpassage, resulting in >90% purity based upon FITC-CD3 mAb staining byflow cytometry. AICD was induced as previously reported¹⁰³. Briefly, Tcells were incubated in RPMI 1640+10% FCS at 2×10⁶/mL and primed with 10μg/ml ConA (Sigma, St. Loius, Mo.) for 48 hrs. Cells were then washedand rested in medium containing 20 U/mL IL-2 (R&D Systems) for 24 hrs.Finally, cells were washed and resuspended in medium containing 20 U/mLIL-2 and increasing amounts of anti-CD3 mAb (0-10 μg/ml) for 24 hrs.Cells were subsequently fixed, stained with 25 μl of 24 μg/mlbis-benzimide trihydrochloride solution (Hoechst #33258; Sigma-Aldrich,Milwaukee Wis.) and apoptosis was quantified by fluorescence microscopy,as above.

Mixed Lymphocyte Reactions (MLR)

Balb/c splenocytes were in vitro activated by incubation with 2×10⁶irradiated (2000 rad) C57BL/6 splenocytes in RPMI 1640+10% FCS for 5days. 2 days prior to the MLR, asmase^(+/+) or asmase^(−/−) splenocyteswere activated with 10 μg/mL con A for 48 hrs, labeled for 45 min with 1μCi/mL Na₂ ⁵¹Cr0₄ at 37° C. and 5% CO₂, washed in RPMI 1640+10% FBS, andcoincubated with activated Balb/c splenocytes in complete medium for 24hrs at 37° C. Target cell lysis was quantified by counting ⁵¹Cr releaseinto the supernatant by gamma counter (Cobra, Meriden, Conn.) using theformula corrected % lysis=100×(sample ⁵¹Cr release−control ⁵¹Crrelease)/(maximum ⁵¹Cr release−control ⁵¹Cr release).

Statistics

Actuarial survival of animals was calculated by the product limitKaplan-Meier method¹⁰⁴ and statistical significance of differences insurvival were calculated by the Mantel log-rank test¹⁰⁵. Crypt survivalcurves were calculated by least square regression analysis, using amodification of the FIT software program⁹⁶. The program fits the curvesby iteratively weighted least squares to each set of dose-survival data,estimates the covariates of the survival curve parameters and thecorresponding confidence regions, and plots the survival curve. It alsoderives curve parameters, such as the D_(o) (the reciprocal of the slopeon the exponential portion of the curve, representing the level ofradiosensitivity) and the N number (measuring the size of the shoulder).Statistical analysis of GVHD scores, thymocyte and splenocyte number,and proliferation assays was performed using the nonparametric unpairedMann-Whitney U test. Student's t-test with 95% confidence estimationswas used for all other analyses.

Example 2 Role of Asmase in GVHD

Small intestine, liver and skin were harvested from asmase^(+/+) andasmase^(−/−) recipients 21 days following transplantation of BM with orwithout 3×10⁶ T cells. Hematoxylin & Eosin stained liver sectionsrevealed hepatic GvHD, characterized by lymphocyte infiltration (FIG.22A, arrows), portal tract inflammation, endotheliitis (FIG. 22A, rightpanels) and loss of hepatic architecture was less prominent inasmase^(−/−) compared to asmase^(+/+) recipients. Similarly, intestinalGvHD, including villus blunting, lamina propria inflammation, crypt stemcell loss and destruction and mucosal atrophy were less prominent inasmase^(−/'1) recipients (FIG. 22B, arrows indicate apoptotic cells).Semiquantitative histopathologic analyses revealed that asmase^(+/+)recipients of allogeneic bone marrow and T cells scored significantlyhigher than littermates receiving only BM in liver (Table 1, 15.7±1.5vs. 8.3±2.7, p<0.05) and small intestine (Table 2, 10.7±1.1 vs. 3.5±0.5,p<0.01). ASMase deficiency largely protected GvHD-associated organdamage, decreasing scoring to 10.2±0.5 and 7±0.1 in liver and smallintestines, respectively (Table 2, p<0.005 each for liver and intestinevs. asmase^(+/+) littermates).

GvHD-associated organ injury is associated with prominent intestine andskin apoptosis. TUNEL-stained ileum sections revealed prominentincidence of apoptotic cells within the lamina propria (FIG. 22C) andcrypt epithelium (FIG. 22D) of asmase^(+/+) recipients of BM and Tcells. asmase^(+/+) recipients of allogeneic T cells exhibited massiveapoptosis (>4 apoptotic cells per villus) in 88.4% of villi, which wasdecreased to 25.4% in asmase^(−/−) recipients (FIG. 22C, p<0.05) and3.8±0.4 apoptotic epithelial cells per crypt, attenuated to 0.95±0.2 inasmase^(−/−) recipients (FIG. 22D, p<0.05). Further, ASMase deficiencyprotected hosts from cutaneous keratinocyte apoptosis following minorantigen-mismatched allogeneic BMT (FIG. 22E). Allogeneic T cells induced5.1±0.9 apoptotic cells/mm in asmase^(−/−) epidermis, compared to8.2±2.1 apoptotic cells/mm of asmase^(+/+) epidermis (p<0.05). Thesedata demonstrate that ASMase tissue are resistant to GvH-associatedapoptosis and organ injury, showing that ASMase regulates GvH-inducedmorbidity and mortality by determining target organ injury andapoptosis. To confirm a specific role for host ASMase in GvHD, a majorhistocompatability-incompatible allogeneic BM transplantation model ofB10.BR donor (H-2^(k)) into C57BL/6 recipient (H-2^(b)) was selected.Lethally-irradiated C57BL/6 hosts of ASMase^(+/+) or ASMase^(−/−)background received 10×10⁶ T-cell depleted (TCD) B 10.BR BM cells, andGvHD was induced by the addition of 0.5×10⁶B10.BR donor splenic T cellsto the allograft. Consistent with the minor-mismatched model,significantly less target organ damage was observed in asmase^(−/−)hosts compared to asmase^(+/+) littermates 14 days followingtransplantation of BM and T cells (Table 1, liver pathology scores of16.3±1.1 in asmase^(+/+) vs. 7.4±0.9 in asmase^(−/'1) hosts, p<0.05, andsmall intestine pathology scores of 10.0±0.8 in asmase^(+/+) vs. 3.5±0.4in asmase^(−/'1) hosts, p<0.05). These data are consistent withdiminished crypt epithelium apoptosis (59.3±3.8% of crypts containingapoptotic cells in wild type vs. 15.8±3.7% in asmase^(−/−) hosts,p<0.05, not shown) and reduced hepatic lymphocyte infiltration,endotheliitis and overall destruction of the hepatic architecture (notshown). Further, keratinocyte apoptosis was prominent in wild typehosts, reaching an apoptotic index of 11.2±1.2 apoptotic cells/mm²epidermis that was significantly attenuated in asmase^(−/−) hosts to3.2±1.7 (FIG. 22E, p<0.01). However, Kaplan-Meier survival could not bedetermined in this major mismatched model due to a late (>day 21 postBMT) BM aplasia, accredited to a genetic shift in the B10.BR straindetected and described on the Jackson laboratory website. Takentogether, these data identify a significant attenuation ofGvHD-associated target organ damage and apoptosis, closely correlatingwith protection against GvH morbidity and mortality, in asmase^(−/−)hosts compared to wild type littermates across both minor and majorantigen disparities.

TABLE 2 Host asmase^(−/−) mediates graf t-vs.-host target organ injuryand apoptosis. Minor-Mismatch Major-Mismatch Model (LP→B6) Model(B10→B6) Transplant Liver Liver Liver Small Intestine BM 8.3 ± 2.7 8.3 ±2.7 1.0 ± 0.0 2.0 ± 0.0 (asmase^(+/+) hosts) BM 7.1 ± 0.9 7.1 ± 0.9 0.7± 0.3 1.5 ± 0.5 (asmase^(−/−) hosts) BM + T 15.7 ± 1.5* 15.7 ± 1.5* 16.3± 1.1* 10.0 ± 0.8* (asmase^(+/+) hosts) BM + T  10.2 ± 0.5**  10.2 ±0.5**  7.4 ± 0.9** 3.5 ± 0.4 (asmase^(−/−) hosts) C57BL/6 recipienthosts received LP BM and T cells (minor mismatc h) as described in FIG.1, or alternatively, received 10 × 10⁶ BM and 0.5 × 10⁶ T cells fromB10.BR donors (major mismatch). asmase^(+/+) and asmase^(−/−) recipientswere sacrificed 14 (B10. BR recipients) or 21 (LP recipients) days postBMT, and t issues were harvested for histopathologic analysis. Smallintestine, large intestine an d liver were scored for establishedorgan-specific parameters in a blinded fashion (mean ± SEM) for 4-14animals per group. *indicates p < 0.01 vs. BM group (asmase^(+/+)hosts), **indicates p < 0.01 vs. BM + T (asmase^(+/+) hosts).

TABLE 3 Table 3. Host asmase^(−/−) regulates graft-vs.-host-inducedserum cytokine expansion. BM BM BM + T BM + T asmase^(+/+) asmase^(−/−)asmase+/+ asmase^(−/−) hosts hosts hosts hosts Minor-Mismatch Model(LP→B6) IL-2 (pg/ml) 15.7 ± 4.1  13.0 ± 1.6  30.3 ± 2.8* 19.1 ± 2.0** IFN-γ (pg/ml) 3.7 ± 1.0 1.3 ± 0.4 109.9 ± 18.9* 32.8 ± 12.4** IL-1β(fg/ml) 6.8 ± 1.9 5.5 ± 0.5 13.4 ± 1.1* 2.9 ± 1.2** TNF-α (pg/ml) 2.3 ±1.2 5.2 ± 1.0 24.3 ± 7.1* 17.3 ± 3.0   Major-Mismatch Model (B.10→B6)IFN-γ (pg/ml) 4.1 ± 1.0 3.6 ± 1.9 199.1 ± 14.2* 63.3 ± 10.8** TNF-α(pg/ml) 33.0 ± 1.9  16.7 ± 2.3  136.8 ± 11.5* 63.7 ± 9.0**  Host C57BL/6were transplantated with LP/J TCD-BM with or without splenic T cells asdescribed in Example 1 and serum was obtained from peripheral blood onday 7 post marrow transplant by retro-orbital puncture. Serum Th1 (IL-2and IFN-γ) and Th2 (IL-1β and TNF-α) cytokines levels were quantified byELISA, as described in Example 1. Serum cytokine levels (mean ± SEM)compiled from 3-8 determinations from three independent experiments foreach group are depicted. *p < 0.005 vs. BM; **p < 0.01 vs. asmase^(+/+).

Example 3 ASMase Deficiency Protects Against Inflammation

We next conducted experiments showing that host ASMase inactivationattenuates Th1/Th2 cytokine profile and CD8⁺ T cell proliferation inacute GvHD. Initial CTL-mediated tissue damage propagates a feed-forwardresponse requiring CD4⁺ Th1 cytokine secretion and consequentalloreactive CD8⁺ clonal expansion for acute GvHD to proceed, followedby inflammatory cytokine storm. To assess the impact of ASMase on serumcytokine levels during GvHD, serum Th1 cytokines IL-2 And IFN-γ and Th2cytokines IL-1β and TNF-α were quantified 7 and 14 days followingtransplantation of LP BM with or without splenic T cells. In the minormismatched model, addition of T cells to the allograft increased IL-2and IFN-γ from 15.7±4.1 to 30.3±2.8 and 3.7±1.0 to 109.9±18.9 pg/mlserum, respectively (p<0.05 for each) on day 7 compared to BM controlrecipients (Table 3). Th2 cytokines IL-1β and TNF-α similarly increasedfrom 6.8±1.9 to 13.4±1.1 and 2.3±1.2 to 24.3±7.0 pg/ml serum,respectively (p<0.05 for each) on day 7 compared to BM controlrecipients (Table 3). Genetic inactivation of host ASMase reduced serumIL-2 and IFN-γ to 19.1±2.0 and 32.8±12.4 pg/ml serum, respectively,(p<0.05 vs. asmase^(+/+) hosts) and IL-1β and TNF-α to 2.9±1.2 and17.3±3.0 pg/ml serum, respectively, (p<0.05 vs. asmase^(+/+) hosts andnot significant) on day 7 (Table 3). Attenuation of serum cytokines waspreserved across major mismatched model of donor B10.BR BM and T cellsinto C57BL/6 hosts (Table 2, 199.1±14.2 vs. 63.3±10.8 pg/ml IFN-γ inasmase vs. asmase^(−/−) hosts p<0.05, 136.8±11.5 vs. 63.7±9.0 pg/mlTNF-α in asmase^(+/+) vs. asmase^(−/−) hosts p<0.005), demonstratingthat ASMase deficiency generally attenuated serum cytokine production inrecipients of allogeneic BM and T cells.

Increased serum cytokine levels directly impact T cell expansion¹¹¹. Toassess whether attenuation of serum cytokines in asmase^(−/'1)recipients of BM and T cells impacts donor T cell proliferation,carboxyl fluorescein succinimidyl ester (CFSE)-labeled T cells(10-20×10⁶) from LP/J (H-2^(b), Minor MHC-incompatible) or B10.BR(H-2¹’, Major MHC-incompatible) donors were infused intolethally-irradiated C57BL/6 hosts of asmase^(+/+) or asmase^(−/−)background. While in vivo expansion of both LP and B10.BR CD4⁺ T cellswere not statistically different in asmase^(−/−) compared toasmase^(+/+) littermates (FIG. 23A upper panels and not shown forB10.BR), CD8⁺ T cell proliferation was significantly impaired acrossboth minor (FIG. 12A) and major MHC (not shown) disparate models. Threedays following infusion, proliferating CD8⁺ cells constituted 54.1% ofthe total donor LP CD8⁺ T cell population in asmase^(+/+) hosts,compared to 26.4% of the population recovered from asmase^(−/'1) hosts(FIG. 23A lower panels, p<0.005). Similarly, proliferating donor B 10.BRCD8⁺ cells were reduced from 81.5% of the total population inasmase^(+/+) hosts to 67.1% in asmase^(−/−) hosts (not shown, p<0.005).Further, attenuated proliferation resulted at 14 days following infusionof BM and 3 ×10⁶ T cells in significant reduction of splenic donor LP/JCD8⁺ T cells from 1.69±0.28 ×10⁶ cells in asmase^(+/+) recipients to0.55±0.28 ×10⁶ in asmase^(−/−) recipients, (FIG. 23B, p<0.001).

Despite impairment of proliferation in vivo, T cell proliferativecapacity remained intact upon transplantation into asmase^(−/−) hosts,as splenic T cells from asmase^(−/−) or asmase^(+/+) hosts allograftedwith LP/J TCD-BM and T cells displayed similar specific proliferativeresponses ex vivo when challenged with conA (FIG. 24). T cellproliferative capacity remains intact in asmase^('1/'1) hosts. Thymidineincorporation assay measuring proliferation of splenic T cells harvestedfrom C57BL/6^(asmase+/+) or C57BL/6^(asmase−/−) recipients of donor LPBM and T cells in response to syngeneic (LP) or allogeneic (Balb/c)splenocytes or mitogen (ConA). Data (mean±SEM) represent triplicatedeterminations from three independent experiments).

Further, ex vivo alloactivation of T cells derived from asmase^(+/+) andasmase^(−/−) hosts 21 days post transplantation with LP/J BM+T cellswere similar, as proliferation was intact in response to irradiatedthird party T cell challenge (Balb/c), and absent in response toirradiated LP/J T cells (FIG. 23). These data demonstrate deficient invivo CD8⁺ CTL proliferation in asmase^('1/'1) hosts, and show abiologically-relevant consequence to the attenuation of serumproinflammatory cytokine levels in these BMT recipients.

Example 4 Methods for Making 2A2 Antibody Hybridoma Generation

C57BL/6 mice underwent eight immunizations with Kaposi' s Sarcoma cells,and serum was collected for evaluation by ELISA. Alternatively, micewere immunized with BSA-conjugated C₁₆-ceramide. Serum was tested forceramide-binding by ELISA, and animals testing positive were sacrificed.Peripheral lymph nodes were harvested and macerated to release B cells.PEG mediated fusion was performed on the purified B cells using Sp2/0myeloma cells, and the fusions were plated in culture plates containingHAT-supplemented DMEM. Hybridomas were subsequently fed on day 5, andsupernatants were collected on day 10 and tested for ceramide binding byELISA. Clones were determined positive only if supernantants boundceramide but not BSA or C₁₆-dihydroceramide. Positive clones wereexpanded in HT-supplemented DMEM, subcloned by limiting dilution, andre-evaluated by ELISA.

ELISA Assay

Antigen (BSA-conjugated C₁₆ ceramide, BSA-conjugated C₁₆ dihydroceramideor BSA) was absorped onto a NUNC Maxisorp Immunoplate by incubationovernight at 4° C. Unbound antigen was washed by phosphate bufferedsaline (PBS) containing 0.05% Tween20, and plates were saturated withPBS containing 5% milk. Hybridoma supernatant or primary antibodyincubation was performed for 2 hrs at room temperature. Following 3washes in PBS+0.05% Tween20, secondary antibody incubation(HRP-conjugated anti-mouse antibody) was performed for 2 hrs at roomtemperature. Following an additional 3 washes, antibody binding wasdetected using 3,3′,5,5′-tetramethylbenzidine (TMB), stopped using anacidic solution and absorbance was quantified at 450 nm.

Antibody Purification

Tissue culture supernatants from hybridomas were sterile filtered andmixed with pre-washed protein G for 1 hr at 4° C. Protein G wascollected and washed with 10 volumes of PBS. Antibodies were eluted with50 mM citrate, 140 mM NaCL, pH 2.7. Antibody containing fractions wereneutralized by Tris, dialyzed and antibody was purified by columnaffinity. Antibody was eluted using NaCL gradient. Antibody was dialyzedagainst 25 mM phosphate, 100 mM NaCL, pH 5.8, aliquoted at 1 mg/ml andstored at -20° C.

Methods for Making 2A2 Antibody Generation of Monoclonal Antibodies.

Five 8-week-old female BALB/c mice were immunized with 0.5 ml of KaposiSarcoma cells (4×10′ /ml) via IP. The animals were immunized threetimes, once per week. Mice were bled 1 week after the last immunization,and the antibody responses in the antiserum were evaluated by FACSanalysis. One mouse was chosen for hybridoma preparation. It received anadditional boost of KS cells three days before the cell fusion.Splenocytes from the BALB/c mice immunized with KS cells were fused withmouse myeloma cells (P3X63Ag8.653, ATCC) at a 4:1 ratio usingpolyethylene glycol (MW 1500). After fusion, cells were seeded andcultured in 96-well plates at 1×10⁵ cells/well in the RPMI 1640selection medium containing 20% fetal bovine serum, 10% Hybridomasupplements (Sigma), 2mM L-glutamine, 100U/ml penicillin, 100 mg/mlstreptomycin, 10 mM HEPES, and 1× hypoxanthine-aminopterin-thymidine(Sigma). Hybridoma supernatants were screened by FACS analysis on KScells and ELISA using commercial available proteins mixture (Annexin V,Ceramide-BSA and APA). Selected hybridomas were subcloned four times bylimited dilution and screened by ELISA on Annexin V coated plates.Conditional media were harvested from the stable hybridoma cultures. TheIg class of mAb was determined with a mouse mAb isotyping kit (SantaCruz). 2A2 mIgM was purified from ascites by using an immobilized mannanbinding protein beads. 2A2 mAb (mIgM) that bind to Annexin V has weakcross-binding activity to Ceremide was confirmed by ELISA.

Antibody Humanization. General molecular cloning techniques (RT-PCR or5′-RACE) are used to obtain the variable regions for both heavy (V_(H))and light (V_(L)) chains of a murine 2A2 antibody. A chimeric 2A2antibody will be generated to confirm binding properties equivalent tothat of the murine parent. Chimeric 2A2 antibody will be humanized byusing complementarity determining region (CDR) grafting. The humanframework sequences from the set of human germ line genes will be chosenbased on similarity of the human antibody's CDRs to the mouse 2A2antibody's CDRs. The antibody will be further engineered to fine tunethe structure of the antigen-binding loops by replacing key residues inthe framework regions of the antibody variable regions to improveantibody affinity and production.

What is claimed is:
 1. A method for preventing or treating a graftversus host disease, comprising administering a therapeuticallyeffective amount of an anti-ceramide antibody or biologically activefragment thereof.
 2. The method of claim 1, wherein the antibody ishumanized.
 3. The method of claim 2, wherein the antibody is amonoclonal antibody.
 4. The method of claim 3, wherein the monoclonalantibody is 2A2 IgM.
 5. A method for preventing or treating a radiationdisease and GI syndrome, comprising administering a therapeuticallyeffective amount of an anti-ceramide antibody or biologically activefragment thereof.
 6. The method of claim 4, wherein the antibody is amonoclonal antibody.
 7. The method of claim 5, wherein the monoclonalantibody is 2A2 IgM.
 8. The method of claim 4, wherein the antibody ishumanized.
 9. A method for preventing or treating an autoimmune disease,comprising administering a therapeutically effective amount of ananti-ceramide antibody or biologically active fragment thereof.
 10. Themethod of claim 9, wherein the antibody is a monoclonal antibody. 11.The method of claim 9, wherein the monoclonal antibody is 2A2 IgM. 12.The monoclonal antibody 2A2 IgM or biologically active fragment thereof.13. The 2A2 IgM of claim 12, wherein the antibody is humanized.
 14. Apharmaceutical composition comprising a humanized anti-ceramide antibodyor biologically active fragment thereof.
 15. A pharmaceuticalcomposition comprising the monoclonal antibody 2A2 IgM or biologicallyactive fragment thereof.
 16. The pharmaceutical composition of claim 14or claim 15, wherein the antibody is humanized.
 17. The pharmaceuticalcomposition of claim 14, further comprising a statin.
 18. Thecomposition of claim 14, further comprising imipramine.
 19. A method forpreventing or treating a radiation disease or GI syndrome, comprisingadministering a therapeutically effective amount of imipramine.
 20. Amethod for preventing or treating graft versus host disease, comprisingadministering a therapeutically effective amount of imipramine.
 21. Amethod for preventing or treating an autoimmune disease, comprisingadministering a therapeutically effective amount of imipramine.
 22. Amethod for treating or preventing graft versus host disease in ananimal, comprising administering a therapeutically effective amount ofan antisense nucleic acid from 8-50 nucleotides in length wherein theantisense nucleic acid is sufficiently complementary to the human geneidentified as SEQ ID NO: 7 or the cDNA identified as SEQ ID NO: 2encoding ASMase to hybridize to it thereby forming a stable duplex. 23.A method for treating or preventing graft versus host disease in ananimal, comprising administering a therapeutically effective amount ofan antisense nucleic acid from 8-50 nucleotides in length wherein theantisense nucleic acid is sufficiently complementary to the human geneidentified as SEQ ID NO: 8 or the cDNA identified as SEQ ID NO: 4encoding encoding Bax to hybridize to it thereby forming a stableduplex.
 24. A method for treating or preventing graft versus hostdisease in an animal, comprising administering a therapeuticallyeffective amount of an antisense nucleic acid from 8-50 nucleotides inlength wherein the antisense nucleic acid is sufficiently complementaryto the human gene identified as SEQ ID NO: 9 or the cDNA identified asSEQ ID NO: 6 encoding encoding Bak to hybridize to it thereby forming astable duplex.
 25. A method for treating or preventing radiation damageor GI syndrome in an animal, comprising administering a therapeuticallyeffective amount of an antisense nucleic acid from 8-50 nucleotides inlength wherein the antisense nucleic acid is sufficiently complementaryto the human gene identified as SEQ ID NO: 7 or the cDNA identified asSEQ ID NO: 2 encoding ASMase to hybridize to it thereby forming a stableduplex.
 26. A method for treating or preventing radiation damage or GIsyndrome in an animal, comprising administering a therapeuticallyeffective amount of an antisense nucleic acid from 8-50 nucleotides inlength wherein the antisense nucleic acid is sufficiently complementaryto the human gene identified as SEQ ID NO: 8 or the cDNA identified asSEQ ID NO: 4 encoding encoding Bax to hybridize to it thereby forming astable duplex.
 27. A method for treating or preventing radiation damageor GI syndrome in an animal, comprising administering a therapeuticallyeffective amount of an antisense nucleic acid from 8-50 nucleotides inlength wherein the antisense nucleic acid is sufficiently complementaryto the human gene identified as SEQ ID NO: 9 or the cDNA identified asSEQ ID NO: 6 encoding encoding Bak to hybridize to it thereby forming astable duplex.
 28. A method for treating or preventing an autoimmunedisease in an animal, comprising administering a therapeuticallyeffective amount of an antisense nucleic acid from 8-50 nucleotides inlength wherein the antisense nucleic acid is sufficiently complementaryto the human gene identified as SEQ ID NO: 7 or the cDNA identified asSEQ ID NO: 2 encoding ASMase to hybridize to it thereby forming a stableduplex.
 29. A method for treating or preventing an autoimmune disease inan animal, comprising administering a therapeutically effective amountof an antisense nucleic acid from 8-50 nucleotides in length wherein theantisense nucleic acid is sufficiently complementary to the human geneidentified as SEQ ID NO: 8 or the cDNA identified as SEQ ID NO: 4encoding encoding Bax to hybridize to it thereby forming a stableduplex.
 30. A method for treating or preventing an autoimmune disease inan animal, comprising administering a therapeutically effective amountof an antisense nucleic acid from 8-50 nucleotides in length wherein theantisense nucleic acid is sufficiently complementary to the human geneidentified as SEQ ID NO: 9 or the cDNA identified as SEQ ID NO: 6encoding encoding Bak to hybridize to it thereby forming a stableduplex.
 31. A method for treating inflammation, comprising administeringa therapeutically effective amount of an anti-ceramide antibody orbiologically active fragment thereof.
 32. The method of claim 5, whereinthe compound is administered before the patient is given radiation. 33.The method of claim 1, wherein the compound is administered before thepatient receives a bone marrow transplant.
 34. A monoclonal antibody orfragment or variant thereof selected from the group comprising 1H4, 15D9 and 5H9 mouse monoclonal antibodies.
 35. The monoclonal antibodiesof claim 34, wherein the antibodies are humanized.
 36. Monoclonalantibodies that cross-react with ceramide, wherein the antibodies areobtained by immunizing the host with whole cells.
 37. The monoclonalantibodies of claim 35, wherein the whole cells are Kaposi Sarcoma (KS)cells or other cells that recapitulate activated endothelium.